Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection

ABSTRACT

The invention provides methods and processes for the identification of polymorphisms at one or more designated sites, without interference from non-designated sites located within proximity of such designated sites. Probes are provided capable of interrogation of such designated sites in order to determine the composition of each such designated site. By the methods of this invention, one or more mutations within the CFTR gene and the HLA gene complex can be can be identified.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/329,427 filed Oct. 14, 2001, U.S. ProvisionalApplication Serial No. 60/329,620, filed Oct. 15, 2001, U.S. ProvisionalApplication Serial No. 60/329,428, filed Oct. 14, 2001 and U.S.Provisional Application Serial No. 60/329,619 filed Oct. 15, 2001. Thisapplication is related to PCT application Serial Number PCT/US02/xxxx ofthe same title filed concurrently herewith. All the above-referencedapplications are expressly incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention generally relates to molecular diagnosticsand genetic typing or profiling. The invention relates to methods,processes and probes for the multiplexed analysis of highly polymorphicgenes. The invention also relates to the molecular typing and profilingof the Human Leukocyte Antigen (HLA) gene complex and the CysticFibrosis Conductance Trans-membrane Regulator gene (CFTR) and tocompositions, methods and designs relating thereto.

BACKGROUND OF THE INVENTION

[0003] The ability to efficiently, rapidly and unambiguously analyzepolymorphisms in the nucleic acid sequences of a gene of interest playsan important role in the development of molecular diagnostic assays, theapplications of which includes genetic testing, carrier screening,genotyping or genetic profiling, and identity testing. For example, itis the objective of genetic testing and carrier screening to determinewhether mutations associated with a particular disease are present in agene of interest. The analysis of polymorphic loci, whether or not thesecomprise mutations known to cause disease, generally provides clinicalbenefit, as for example in the context of pharmacogenomic genotyping orin the context of HLA molecular typing, in which the degree of allelematching in the HLA loci of transplant donor and prospective recipientis determined in context of allogeneic tissue and bone marrowtransplantation.

[0004] The multiplexed analysis of polymorphisms while desirable infacilitating the analysis of a high volume of patient samples, faces aconsiderable level of complexity which will likely increase as newpolymorphisms, genetic markers and mutations are identified and must beincluded in the analysis. The limitations of current methods to handlethis complexity in a multiplexed format of analysis so as to ensurereliable assay performance while accommodating high sample volume, andthe consequent need for novel methods of multiplexed analysis ofpolymorphisms and mutations is the subject of the present invention. Byway of example, the genetic loci encoding Cystic Fibrosis TransmembraneConductance (CFTR) channel and Human Leukocyte Antigens (HLA) areanalyzed by the methods of the invention. Cystic fibrosis (CF) is one ofthe most common recessive disorders in Caucasians with a rate ofoccurrence in the US of 1 in 2000 live births. About 4% of thepopulation carry one of the CF mutations. The CFTR gene is highlyvariable: more than 900 mutations have been identified to date (seehttp://www.genet.sickkids.on.ca/cftr, which is incorporated herein byreference). The characterization of the CFTR gene provides the key tothe molecular diagnosis of CF by facilitating the development ofsequence-specific probes (Rommens et al., 1989; Riordan, et al., 1989;Kerem et al., 1989, each of which is incorporated herein by reference).The National Institutes of Health (NIH)—sponsored consensus developmentconference recommended carrier screening for CFTR mutations for adultswith a positive family history of CF (NIH 1997). The committee oncarrier screening of the American College of Medical Genetics (ACMG) hasrecommended for use in general population carrier screening a pan-ethnicmutation panel that includes a set of 25 disease-causing CF mutationswith an allele frequency of >0.1% in the general population of UnitedStates (see http://www.faseb.org/genetics/acmg, which is incorporatedherein by reference). The mutations in the ACMG panel also include themost common mutations in Ashkenazi Jewish and African-Americanpopulations.

[0005] Several methods have been described for the detection of CFTRmutations including the following: : denaturing gradient gelelectrophoresis (Devoto et al., 1991); single strand conformationpolymorphism analysis (Plieth et al., 1992); RFLP (Friedman et al.,1991); amplification with allele-specific primers (ASPs) (Gremonesi etal., 1992), and probing with allele specific oligonucleotides (ASO)(Saiki et al., 1986). A widely used method involves PCR amplificationfollowed by blotting of amplified target strands onto a membrane andprobing of strands with oligonucleotides designed to match either thenormal (“wild type”) or mutant configuration. Specifically, multiplexPCR has been used in conjunction with ASO hybridization in this dot blotformat to screen 12 CF mutations (Shuber et al., 1993). In severalinstances, arrays of substrate-immobilized oligonucleotide probes wereused to facilitate the detection of known genomic DNA sequencevariations (Saiki, R K et al., 1989) in a “reverse dot blot” format Anarray of short oligonucleotides synthesized in-situ by photolithographicprocesses was used to detect known mutations in the coding region of theCFTR gene (Cronin, M T., et al., 1996). Primer extension using reversetranscriptase has been reported as a method for detecting the Δ508mutation in CFTR (Pastinen, T., 2000). This approach was described asearly as 1989 (Wu, D. Y. et al, Proc. Natl. Acad. Sci. USA. 86:2757-2760(1989), Newton, C. R. et al, Nucleic Acids Res. 17:2503-2506 (1989)). Asfurther discussed herein below, while providing reasonable detection ina research laboratory setting, these methods require significant labor,provide only slow turnaround, offer only low sample throughput, andhence require a high cost per sample.

[0006] In connection with the spotted microarrays, several methods ofspotting have been described, along with many substrate materials andmethods of probe immobilization. However, the spotted arrays of currentmethods exhibit not only significant array-to-array variability but alsosignificant spot-to-spot variability, an aspect that leads tolimitations in assay reliability and sensitivity. In addition, spottedarrays are difficult to miniaturize beyond their current spot dimensionsof typically 100 μm diameter on 500 μm centers, thereby increasing totalsample volumes and contributing to slow assay kinetics limiting theperformance of hybridization assays whose completion on spotted arraysmay require as much as 18 hours. Further, use of spotted arrays involvereadout via highly specialized confocal laser scanning apparatus. In analternative approach, oligonucleotide arrays synthesized in-situ by aphotolithographic process have been described. The complexity of arrayfabrication, however, limits routine customization and combinesconsiderable expense with lack of flexibility for diagnosticapplications.

[0007] The major histocompatibility complex (MHC) includes the humanleukocyte antigen (HLA) gene complex, located on the short arm of humanchromosome six. This region encodes cell-surface proteins which regulatethe cell-cell interactions underlying immune response. The various HLAClass I loci encode 44,000 dalton polypeptides which associate with β-2microglobulin at the cell surface and mediate the recognition of targetcells by cytotoxic T lymphocytes. HLA Class II loci encode cell surfaceheterodimers, composed of a 29,000 dalton and a 34,000 daltonpolypeptide which mediate the recognition of target cells by helper Tlymphocytes. HLA antigens, by presenting foreign pathogenic peptides toT-cells in the context of a “self” protein, mediate the initiation of animmune response. Consequently, a large repertoire of peptides isdesirable because it increases the immune response potential of thehost. On the other hand, the correspondingly high degree ofimmunogenetic polymorphism represents significant difficulties inallotransplantation, with a mismatch in HLA loci representing one of themain causes of allograft rejection. The degree of allele matching in theHLA loci of a donor and prospective recipient is a major factor in thesuccess of allogeneic tissue and bone marrow transplantation.

[0008] The HLA-A, HLA-B, and HLA-C loci of the HLA Class I region aswell as the HLA-DRB, HLA-DQB, HLA-DQA, HLA-DPB and HLA-DPA loci of theHLA Class II region exhibit an extremely high degree of polymorphism. Todate, the WHO nomenclature committee for factors of the HLA system hasdesignated 225 alleles of HLA A (HLA A*0101, A*0201, etc.), 444 allelesof HLA-B, and 111 alleles of HLA-C, 358 HLA-DRB alleles, 22 HLA-DQAalleles, 47 HLA-DQB alleles, 20 HLA-DPA alleles and 96 HLA-DPB alleles(See IMGT/HLA Sequence Database,http://www3.ebi.ac.uk:80/imgt/hla/index.html) and Schreuder, G. M. Th.et al, Tissue Antigens. 54:409-437 (1999)), both of which are herebyincorporated by reference.

[0009] HLA typing is a routine procedure that is used to determine theimmunogenetic profile of transplant donors. The objective of HLA typingis the determination of the patient's allele configuration at therequisite level of resolution, based on the analysis of a set ofdesignated polymorphisms within the genetic locus of interest.Increasingly, molecular typing of HLA is the method of choice overtraditional serological typing, because it eliminates the requirementfor viable cells, offers higher allelic resolution, and extends HLAtyping to Class II for which serology has not been adequate (Erlich, H.A. et al, Immunity. 14:347-356 (2001)).

[0010] One method currently applied to clinical HLA typing uses thepolymerase chain reaction (PCR) in conjunction with sequence-specificoligonucleotide probes (SSO or SSOP), which are allowed to hybridize toamplified target sequences to produce a pattern as a basis for HLAtyping.

[0011] The availability of sequence information for all available HLAalleles has permitted the design of sequence-specific oligonucleotides(SSO) and allele-specific oligonucleotides (ASO) for thecharacterization of known HLA polymorphisms as well as for sequencing byhybridization (Saiki, R. K. Nature 324:163-166 (1986), Cao, K. et al,Rev Immunogenetics, 1999: 1: 177-208).

[0012] In one embodiment of SSO analysis, also referred to as a “dotblot format”, DNA samples are extracted from patients, amplified andblotted onto a set of nylon membranes in an 8×12 grid format. Oneradio-labeled oligonucleotide probe is added to each spot on each suchmembrane; following hybridization, spots are inspected byautoradiography and scored either positive (1) or negative (0). For eachpatient sample, the string of l's and 0's constructed from the analysisof all membranes defines the allele configuration. A multiplexed formatof SSO analysis in the “reverse dot blot format” employs sets ofoligonucleotide probes immobilized on planar supports (Saiki, R. et al,Immunological Rev. 167: 193-199 (1989), Erlich, H. A. Eur. J.Immunogenet. 18: 33-55 (1991)).

[0013] Another method of HLA typing uses the polymerase-catalyzedelongation of sequence-specific primers (SSPs) to discriminate betweenalleles. The high specificity of DNA polymerase generally endows thismethod with superior specificity. In the SSP method, PCR amplificationis performed with a specific primer pair for each polymorphic sequencemotif or pair of motifs and a DNA polymerase lacking 3′->5′ exonucleaseactivity so that elongation (and hence amplification) occurs only forthat primer whose 3′ terminus is perfectly complementary (“matched”) tothe template. The presence of the corresponding PCR product isascertained by gel electrophoretic analysis. An example of a highlypolymorphic locus is the 280 nt DNA fragment of the HLA class II DR genewhich features a high incidence of polymorphisms

[0014] HLA typing based on the use of sequence-specific probes (SSP),also referred to as phototyping (Dupont, B. Tissue Antigen. 46: 353-354(1995)), has been developed as a commercial technology that is inroutine use for class I and class II typing (Bunce, M. et al, TissueAntigens. 46:355-367 (1995), Krausa, P and Browning, M. J., TissueAntigens. 47: 237-244 (1996), Bunce, M. et al, Tissue Antigens. 45:81-90(1995)). However, the requirement of the SSP methods of the prior artfor extensive gel electrophoretic analysis for individual detection ofamplicons represents a significant impediment to the implementation ofmultiplexed assay formats that can achieve high throughput. Thisdisadvantage is overcome by the methods of the present invention.

[0015] In the context of elongation reactions, highly polymorphic lociand the effect of non-designated polymorphic sites as interferingpolymorphisms were not considered in previous applications, especiallyin multiplexed format. Thus, there is a need to provide for methods,compositions and processes for the multiplexed analysis of polymorphicloci that would enable the detection of designated while accommodatingthe presence of no-designated sites and without interference from suchnon-designated sites.

SUMMARY OF THE INVENTION

[0016] The present invention provides methods and processes for theconcurrent interrogation of multiple designated polymorphic sites in thepresence of non-designated polymorphic sites and without interferencefrom such non-designated sites. Sets of probes are provided whichfacilitate such concurrent interrogation. The present invention alsoprovides methods, processes, and probes for the identification ofpolymorphisms of the HLA gene complex and the CFTR gene.

[0017] The specificity of methods of detection using probe extension orelongation is intrinsically superior to that of methods usinghybridization, particularly in a multiplexed format, because thediscrimination of sequence configurations no longer depends ondifferential hybridization but on the fidelity of enzymatic recognition.To date, the overwhelming majority of applications of enzyme-mediatedanalysis use single base probe extension. However, probe elongation, inanalogy to that used in the SSP method of HLA typing, offers severaladvantages for the multiplexed analysis of polymorphisms, as disclosedherein. Thus, single nucleotide as well as multi-nucleotidepolymorphisms are readily accommodated. The method, as described herein,is generally practiced with only single label detection, accommodatesconcurrent as well as consecutive interrogation of polymorphic loci andincorporates complexity in the probe design.

[0018] One aspect of this invention provides a method of concurrentdetermination of nucleotide composition at designated polymorphic siteslocated within one or more target nucleotide sequences. This methodcomprises the following steps: (a) providing one or more sets of probes,each probe capable of annealing to a subsequence of the one or moretarget nucleotide sequences located within a range of proximity to adesignated polymorphic site; (b) contacting the set of probes with theone or more target nucleotide sequences so as to permit formation ofhybridization complexes by placing an interrogation site within a probesequence in direct alignment with the designated polymorphic site; (c)for each hybridization complex, determining the presence of a match or amismatch between the interrogation site and a designated polymorphicsite; and (d) determining the composition of the designated polymorphicsite.

[0019] Another aspect of this invention is to provide a method ofsequence-specific amplification of assay signals produced in theanalysis of a nucleic acid sequence of interest in a biological sample.This method comprises the following steps: (a) providing a set ofimmobilized probes capable of forming a hybridization complex with thesequence of interest; (b) contacting said set of immobilized probes withthe biological sample containing the sequence of interest underconditions which permit the sequence of interest to anneal to at leastone of the immobilized probes to form a hybridization complex; (c)contacting the hybridization complex with a polymerase to allowelongation or extension of the probes contained within the hybridizationcomplex; (d) converting elongation or extension of the probes into anoptical signal; and (e) recording the optical signal from the set ofimmobilized probes in real time.

[0020] Yet another aspect of this invention is to provide a method offorming a covering probe set for the concurrent interrogation of adesignated polymorphic site located in one or more target nucleic acidsequences. This method comprises the steps of: (a) determining thesequence of an elongation probe capable of alignment of theinterrogation site of the probe with a designated polymorphic site; (b)further determining a complete set of degenerate probes to accommodateall non-designated as well as non-selected designated polymorphic siteswhile maintaining alignment of the interrogation site of the probe withthe designated polymorphic site; and (c) reducing the degree ofdegeneracy by removing all tolerated polymorphisms.

[0021] One aspect of this invention is to provide a method foridentifying polymorphisms at one or more designated sites within atarget polynucleotide sequence. This the method comprise the followingsteps: (a) providing one or more probes capable of interrogating saiddesignated sites; (b) assigning a value to each such designated sitewhile accommodating non-designated polymorphic sites located within arange of proximity to each such polymorphism.

[0022] Another aspect of this invention is to provide a method fordetermining a polymorphism at one or more designated sites in a targetpolynucleotide sequence. This method comprises providing a probe set forthe designated sites and grouping the probe set in different probesubsets according to the terminal elongation initiation of each probe.

[0023] Another aspect of this invention is to provide a method for theconcurrent interrogation of a multiplicity of polymorphic sitescomprising the step of conducting a multiplexed elongation assay byapplying one or more temperature cycles to achieve linear amplificationof such target.

[0024] Yet another aspect of this invention is to provide a method forthe concurrent interrogation of a multiplicity of polymorphic sites.This method comprises the step of conducting a multiplexed elongationassay by applying a combination of annealing and elongation steps undertemperature-controlled conditions.

[0025] Another aspect of this invention is to provide a method ofconcurrent interrogation of nucleotide composition at S polymorphicsites, P_(S):={c_(P)(s); 1≦s≦S} located within one or more contiguoustarget sequences, said method assigning to each c_(P) one of a limitedset of possible values by performing the following steps: (a) providinga set of designated immobilized oligonucleotide probes, also known aselongation probes, each probe capable of annealing in a preferredalignment to a subsequence of the target located proximal to adesignated polymorphic site, the preferred alignment placing aninterrogation site within the probe sequence in direct juxtaposition tothe designated polymorphic site, the probes further containing aterminal elongation initiation (TEI) region capable of initiating anelongation or extension reaction ;(b) permitting the one or more targetsequences to anneal to the set of immobilized oligonucleotide probes soas form probe-target hyrbdization complexes; and (c) for eachprobe-target hybridization complex, calling a match or a mismatch incomposition between interrogation site and corresponding designatedpolymorphic site.

[0026] Other objects, features and advantages of the invention will bemore clearly understood when taken together with the following detaileddescription of an embodiment which will be understood as beingillustrative only.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1a is an illustration of probe sets designed to interrogatedesignated sites in HLA-DR and an internal control.

[0028]FIG. 1b is an illustration of a staggered primer design.

[0029]FIG. 2 is an illustration of a modification of allele bindingpattern based on tolerance effect.

[0030]FIG. 3 is an illustration of the use of linked primer structure toseparate the anchoring sequence and polymorphism detection sequence.

[0031]FIG. 4 shows simulated ambiguity in allele identification due toallele combination.

[0032]FIG. 5 shows one method for decreasing the ambiguity in alleleidentification that arises from allele combination.

[0033]FIG. 6 is an illustration of a combination of hybridization andelongation.

[0034]FIG. 7 shows a model reaction using synthetic oligonucleotides astargets.

[0035]FIG. 8 shows results obtained using testing real patient sample inan eMAP format.

[0036]FIG. 9 shows results obtained from eMAP primer extension for DRlocus.

[0037]FIG. 10 shows results obtained from eMAP for DR locus.

[0038]FIG. 11 shows results obtained from eMAP for A locus Exon 3.

[0039]FIG. 12 shows results obtained from eMAP SSP for A locus Exon 3and is an example of tolerance for the non-designated polymorphism.

[0040]FIG. 13 is an illustration of bead immobilized probe elongation ofvariable mutant sites.

[0041]FIG. 14 is an illustration of PCR using primers immobilized on thesurface of beads.

[0042]FIG. 15 is an illustration of elongation of multiple probes usingcombined PCR products.

[0043]FIG. 16 is an illustration of results for probe elongation of amultiplexed CF mutation.

[0044]FIG. 16a is an illustration of probe elongation using a synthetictarget.

[0045]FIG. 16b is an illustration of probe elongation using beads in aPCR reaction.

[0046]FIG. 17 is an illustration of one-step elongation withtemperature-controlled cycling results.

[0047]FIG. 18 is an illustration of primer elongation with labeled dNTPand three other unlabeled dNTPs.

[0048]FIG. 19 is an illustration of primer elongation with labeled ddNTPand three other unlabeled dNTPs.

[0049]FIG. 20 is an illustration of primer elongation, where fourunlabeled dNTPs are used for elongation and the product is detected by alabeled oligonucleotide probe which hybridizes to the extended unlabeledproduct.

[0050]FIG. 21 is an illustration of a primer extension in which alabeled target and four unlabeled dNTPs are added. This illustrationwhich shows that only with the extended product can the labeled targetbe retained with the beads when high temperature is applied to the chip.

[0051]FIG. 22 is an illustration of linear amplification where sequencespecific probes are immobilized.

[0052]FIG. 23 is an illustration of the utilization of hairpin probes.

[0053]FIG. 24 is an illustration of applying this invention to theanalysis of cystic fibrosis and Ashkenazi Jewish disease mutations.

DETAILED DESCRIPTION OF THE INVENTION

[0054] This invention provides compositions, methods and designs for themultiplexed analysis of highly polymorphic loci; that is, loci featuringa high density of specific (“designated”) polymorphic sites, as well asinterfering non-designated polymorphic sites. The multiplexed analysisof such sites thus generally involves significant overlap in thesequences of probes directed to adjacent sites on the same target, suchthat probes designed for any specific or designated site generally alsowill cover neighboring polymorphic sites. The interference in theanalysis of important genes including CFTR and HLA has not beenaddressed in the prior art. To exemplify the methods of the methods ofthe invention, the HLA gene complex and the CFTR gene are analyzed.

[0055] The present invention provides compositions and methods for theparallel or multiplexed analysis of polymorphisms (“MAP”) in nucleicacid sequences displaying a high density of polymorphic sites. In agiven nucleic acid sequence, each polymorphic site comprises adifference comprising one or more nucleotides.

[0056] This invention provides methods and compositions for theconcurrent interrogation of an entire set of designated polymorphismswithin a nucleic acid sequence. This invention provides compositions,methods and designs to determine the composition at each such site andthereby provide the requisite information to select, from the set ofpossible configurations for the sequence of interest, the actualconfiguration in a given specific sample. The invention also serves tonarrow the set of possible sequences in that sample. Accordingly, incertain embodiments, it will be useful or necessary to determinesequence composition by assigning to a designated site one of thepossible values corresponding to nucleotide identity. In otherembodiments, it will be sufficient to determine the site composition tobe either matching or non-matching with respect to a known referencesequence, as in the assignment of “wild-type” or “mutation” in thecontext mutation analysis. The capability of sequence determinationthereby afforded is referred to herein as confirmatory sequencing orresequencing. In a preferred embodiment, the present invention provideselongation-mediated multiplexed analysis of polymorphisms (eMAP) of theCystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and forthe Human Leukocyte Antigen (HLA) gene complex.

[0057] The methods and compositions of this invention are useful forimproving the reliability and accuracy of polymorphism analysis oftarget regions which contain polymorphic sites in addition to thepolymorphic sites designated for interrogation. These non-designatedsites represent a source of interference in the analysis. Depending onthe specific assay applications, one or more probes of differingcomposition may be designated for the same polymorphic site, aselaborated in several Examples provided herein. It is a specificobjective of the present invention to provide compositions and methodsfor efficient, rapid and unambiguous analysis of polymorphisms in genesof interest. This analysis is useful in molecular diagnostic assays,such as those designed, for example, for genetic testing, carrierscreening, genotyping or genetic profiling, identity testing, paternitytesting and forensics.

[0058] Preparation of target sequences may be carried out using methodsknown in the art. In a non-limiting example, a sample of cells or tissueis obtained from a patient. The nucleic acid regions containing targetsequences (e.g., Exons 2 and 3 of HLA) are then amplified using standardtechniques such as PCR (e.g., asymmetric PCR).

[0059] Probes for detecting polymorphic sites function as the point ofinitiation of a polymerase-catalyzed elongation reaction when thecomposition of a polymorphic site being analyzed is complementary(“matched”) to that of the aligned site in the probe. Generally, theprobes of the invention should be sufficiently long to avoid annealingto unrelated DNA target sequences. In certain embodiments, the length ofthe probe may be about 10 to 50 bases, more preferably about 15 to 25,and more preferably 18 to 20 bases. Probes may be immobilized on thesolid supports via linker moieties using methods and compositions wellknown in the art.

[0060] As used herein, the term “nucleic acid” or “oligonucleotide”refers to deoxyribonucleic acid or ribonucleic acid in a single ordouble-stranded form. The term also covers nucleic-acid like structureswith synthetic backbones. DNA backbone analogues include phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs). See Oligonucleotides and Analogues, APractical Approach (Editor: F. Eckstein), IRL Press at Oxford UniversityPress (1991); Antisense Strategies, Annals of the New York Academy ofSciences, vol. 600, Eds.; Baserga and Denhardt (NYAS 1992); Milligan, J.Med. Chem., vol. 36, pp. 1923-1937; Antisense Research and Applications(1993, CRC Press). PNAs contain non-ionic backbones, such asN-2(2-aminoethyl) glycine units. Phosphorothioate linkages are describedin WO 97/0321 1;WO 96/39159; and Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997). Other synthetic backbones encompassed by the terminclude methyl-phosphonate linkages or alternating methylphosphonate andphosphodiester linkages (Strauss-Soukup, Biochemistry, 36: 8692-8698(1997), and benzylphosphonate linkages (Samstag, Antisense Nucleic AcidDrug Dev., 6: 153-156 (1996)). The term nucleic acid includes genes,cDNAs, and mRNAs.

[0061] As used herein, the term “hybridization” refers to the binding,duplexing, or hybridizing of a nucleic acid molecule preferentially to aparticular nucleotide sequence under stringent conditions. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to the corresponding target sequence, and to alesser extent or not at all to other sequences. A “stringenthybridization” is sequence dependent, and is different under differentconditions. An extensive guide to the hybridization of nucleic acids maybe found in, e.g. Tijssen, Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier, N.Y. (1993). Generally, highly stringenthybridization and wash conditions are selected to about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Very stringent conditions are selected byconducting the assay at a temperature set to be equal to the T_(m) for aparticular probe. An example of highly stringent wash condition is 0.15M NaCl at 72° C. for about 15 minutes. An example of stringent washconditions is a 0.2×SSC wash at 65° C. for 15 minutes. See Sambrook,Molecular Cloning: A Laboratory Manual (2^(nd) Ed), vol. 1-3 (1989).

[0062] As used herein, the term “designated site” is defined as apolymorphic site of interest (i.e., a polymorphic site that one intendsto identify) on a given nucleic acid. The term “non-designated site”refers to any polymorphic site that co-exists with a designated site orsites on a given nucleic acid but is not of interest.

[0063] As used herein, the term “correlated designated sites” refers topolymorphic sites with correlated occurrences. Typically, each member ofsuch a set of polymorphic sites must be identified in order to identifythe allele to which the set belongs.

[0064] As used herein, the term “selected designated site” refers to apolymorphic site of interest on a given nucleic acid that also overlapswith the 3′ end of a probe sequence of this invention. A “non-selecteddesignated site” refers to a polymorphic site of interest that does notoverlap with a 3′ end of a probe sequence of this invention.

[0065] As used herein, an “interfering non-designated site” refers to anon-designated polymorphic site that is within 1-5 bases from the 3′ endof a probe sequence of this invention. A “non-interfering non-designatedsite” refers to a non-designated site that is greater than 5 bases fromthe 3′ end of a probe sequence of this invention. The non-interferingnon-designated site may be closer to the 5′ end of the probe sequencethan to the 3′ end.

[0066] In certain embodiments, the probes of this invention comprise a“terminal elongation initiation” region (also referred to as a “TEI”region) and a Duplex Anchoring (“DA”) region. The TEI region refers asection of the probe sequence, typically the three or four 3′ terminalpositions of the probe. The TEI region is designed to align with aportion of the target nucleic acid sequence at a designated polymorphicsite so as to initiate the polymerase-catalyzed elongation of the probe.The DA region, typically comprises the remaining positions within theprobe sequence and is preferably designed to align with a portion of thetarget sequence in a region located close (within 3-5 bases) to thedesignated polymorphism.

[0067] As used herein, the term a “close range of proximity” refers to adistance of between 1-5 bases along a given nucleic acid strand. A“range of proximity” refers to a distance within 1-10 bases along agiven nucleic acid strand. The term “range of tolerance” refers to thetotal number of mismatches in the TEI region of a probe hybridized to atarget sequence that still permits annealing and elongation of theprobe. Typically, more than 2 mismatches in the TEI region of ahybridized probe is beyond the range of tolerance.

[0068] The terms “microspheres”, “microparticles”, “beads”, and“particles” are herein used interchangeably. The composition of thebeads includes, but is not limited to, plastics, ceramics, glass,polystyrene, methylstyrene, acrylic polymers, paramagnetic materials,thoria sol, carbon graphite, titanium dioxide, latex or cross-linkeddextrans such as sepharose, cellulose, nylon, cross-linked micelles andTeflon. See “Microsphere Detection Guide” from Bangs Laboratories,Fishers IN. The particles need not be spherical and may be porous. Thebead sizes may range from nanometers (e.g., 100 nm) to millimeters(e.g., 1 mm), with beads from about 0.2 micron to about 200 micronsbeing preferred, more preferably from about 0.5 to about 5 micron beingparticularly preferred.

[0069] This invention provides for the concurrent interrogation of a setof designated polymorphic sites within one or more target strands byfirst annealing a set of immobilized sequence specific oligonucleotideprobes to target nucleic acid strands and by probing the configurationof designated polymorphic sites by way of polymerase-catalyzedelongation of the annealed set of immobilized sequence-specificoligonucleotide probes. An elongation probe is designed to interrogate adesignated site by annealing to a sequence in a given target, therebyforming a hybridization complex (“duplex”). The probe's 3′ terminus isplaced at or near the designated site within the target andpolymerase-catalyzed probe elongation is initiated if the 3′ terminalprobe composition matches (i.e., is complementary to) that of the targetat the interrogation site. As described herein, the probe may bedesigned to anneal in a manner such that the designated site is within arange of proximity of the 3′ terminus.

[0070] In one embodiment of the invention, two or more probes may beprovided for interrogation of a specific designated site. The probes aredesigned to take into account the possibility of polymorphisms ormutations at the interrogation site and non-designated polymorphic siteswithin a certain range of proximity of the designated polymorphic site.In this context, the term “polymorphism” refers to any variation in anucleic acid sequence, while the term “mutation” refers to a sequencevariation in a gene that is associated or believed to be associated witha phenotype. In a preferred embodiment, this multiplicity of probesequences contains at least one probe that matches the specific targetsequence in all positions within the range of proximity to ensureelongation.

[0071] In certain embodiments, the invention discloses compositions andmethods for the parallel interrogation of S polymorphic sites selectedfrom a target sequence of length N by a set of L≧S oligonucleotideprimers.

[0072] In accordance with the requirements of specific assayapplications, one or more probes of differing composition may bedesignated for the same polymorphic site, as elaborated in severalExamples provided herein.

[0073] Each designated probe is composed of a nucleotide sequence oflength M which contains an interrogation site (one that, uponhybridization, aligns with the polymorphic site being analyzed) at ornear the 3′ terminus. Although 3′ end is preferred, those within 3-4bases from the 3′ end may be used. The primer is immobilized on a solidphase carrier (may be linked via a linker sequence or other linkermoiety) and is identified by its association with that carrier. Theprobe sequence is designed to permit annealing of the primer with thetarget so as to form a hybridization complex between probe and targetand to ensure the alignment of the interrogation site with thedesignated polymorphic site, the preferred configuration providing aninterrogation site at the probe's 3′ terminus and alignment of the 3′terminus with the designated polymorphic site. The step of interrogatingthe nucleotide composition of the designated polymorphic site with adesignated probe of given interrogation site composition assigns to thatsite one of two values, namely matched, numerically represented by 1, ornon-matched, numerically represented by 0. In HLA molecular typing, theresulting binary string of length L identifies an allele to a desiredtyping resolution.

[0074] In a preferred embodiment, the interrogation step uses theextension of the designated probe. This reaction, catalyzed by apolymerase, produces an extended hybridization complex by adding to theprobe sequence one or more nucleoside triphosphates in the orderreflecting the sequence of the target sequence in the existinghybridization complex. In order for this extension reaction to proceed,a designated primer of length M must contain a terminal extensioninitiation region of length M*≦M, herein also referred to as terminalextension initiation sequence (or TEI sequence), which contains theinterrogation site. Extension proceeds if the composition of thedesignated interrogation site matches that of the designated polymorphicsite.

[0075] Methods of the prior art of detecting successful extension havebeen described which involve the use labeled deoxy nucleosidetriphosphates (dNTPs) or dideoxy nucleoside triphosphates (ddNTPs). Thepresent invention also discloses novel methods of providing opticalsignatures for detection of successful extension eliminating the needfor labeled dNTPs or ddNTPs, an advantage arising from the reduction inthe efficiency of available polymerases in accommodating labeled dNTPsor ddNTPs.

[0076] However, the density of polymorphic sites in highly polymorphicloci considered in connection with the present invention makes it likelythat designated primers directed to selected polymorphic sites, whenannealing to the target subsequence proximal to the designatedpolymorphic site, will overlap adjacent polymorphic sites.

[0077] That is, an oligonucleotide probe, designed to interrogate theconfiguration of the target at one of the selected polymorphic sites,and constructed with sufficient length to ensure specificity and thermalstability in annealing to the correct target subsequence, will alignwith other nearby polymorphic sites. These interfering polymorphic sitesmay include the non-designated sites as well as non-selected designatedsites in the target sequence.

[0078] In a multiplexed SSP reaction carried out in solution, thepartial overlap between designated probes directed to nearby selectedpolymorphisms may lead to mutual competition between probes for the sametarget. The present invention significantly reduces this complication byway of probe immobilization.

[0079] As with multiplexed differential hybridization generally, themismatch in one or more positions between a designated probe and targetmay affect the thermal stability of the hybridization complex. That is,any set of annealing conditions applied to the entire reaction mixturemay produce varying degrees of annealing between probe and target andmay affect the outcome of the subsequent probe extension reaction,thereby introducing ambiguities in the assay which may requiresubsequent resequencing.

[0080] Non-designated polymorphic sites located in immediate proximityto the interrogation site near or at the 3′ terminus of the designatedprobe are particularly deleterious to the effectiveness of the probe'sTEI sequence in initiating the extension reaction.

[0081] The power of currently available polymerase enzymes catalyzingthe extension reaction to discriminate between a match and a mismatch incomposition between the interrogation site within the designated primerand the polymorphic site depends on the displacement of theinterrogation site from the primer's 3′ terminus, considering singlenucleotide as well as multiple nucleotide polymorphisms.

[0082] In a preferred embodiment yielding optimal discriminating power,the interrogation site is provided at the probe's 3′ terminus. Given aprobe sequence of length M designated for a selected site s* in therepresentation P_(M(s*)):={c_(P(m)); 1≦m≦M}, the index m increasing inthe primer's 5′ to 3′ direction, this configuration provides foralignment of the designated site s* with position M in the probesequence; in the case of multiple nucleotide polymorphisms, positionsM−1 (for a dinucleotide polymorphism) and M−2 (for a trinucleotidepolymorphism), etc. also are implicated.

[0083] Under these circumstances as they are anticipated in themultiplexed analysis of highly polymorphic loci, the advantage ofenhanced specificity afforded by the application of apolymerase-catalyzed extension reaction is greatly diminished or lost asa result of complications arising from “sub-optimal” annealingconditions closely related to those limiting the performance of SSOanalysis.

[0084] In connection with the optimization of the design of multipleprobe sequences sharing the same interrogation site composition for anygiven designated polymorphic site, it will be useful to consider theconcept of tolerance of interfering polymorphisms. Considering withoutlimitation of generality the example of the single nucleotidepolymorphism, a shift in alignment of s* away from the 3′ terminus topositions M−1, M−2, . . . , M−m* leads to a gradually diminisheddiscriminatory power. That is, when the designated polymorphic site isaligned with an interior probe position, m*, the extension reaction nolonger discriminates between match and mismatch. Conversely, in thepreferred embodiment of placing the interrogation site at the probe's 3′terminus, the deleterious effect of nearby non-designated polymorphismson the effectiveness of the extension reaction likewise decreases withdistance from the 3′ terminus. That is, non-designated polymorphismsaligned with position between 1 and m* will not affect the extensionreaction.

[0085] The terminal sequence of length M−m*+1 within the probe is hereinreferred to as the TEI sequence of a given primer. In general, 1<m*<M,and the TEI sequence may comprise only small number of terminal probepositions; in certain cases, m*=1, so that the probe sequenceencompasses the entire probe sequence.

[0086] The present invention accommodates the presence of interferingpolymorphic sites within the length of a designated probe sequence bytaking into account these known sequence variations in the design ofmultiple probes. In particular, the number of alternate probe sequenceconfigurations to be provided for given probe length M is significantlyreduced as a result of the existence of a TEI sequence of length M−m*+1.That is, in order to ensure effective discriminatory power of theextension reaction, it is sufficient to restrict the anticipatoryalternate probe sequence configurations to the length of the TEIsequence. In a preferred embodiment, all possible alternative sequencesare anticipated so that one of these alternate probe sequences willmatch the target in all of the positions m*, m*+1, . . . M−1, M.

[0087] Providing, for each selected polymorphic site, a multiplicity ofdesignated probes with anticipatory sequences increases the complexityof coding if all of these probes are separately encoded by the uniqueassociation with coded solid phase carriers. However, this complexity isreduced by placing this set of probes on a common solid phase carrier.That is, only the interrogation site composition of any designatedprobes is encoded, a concept herein referred to as TEI sequence poolingor probe pooling. Complete probe sequence pooling reduces the codingcomplexity to that of the original design in which no anticipatory probesequences were provided. Partial pooling also is possible.

[0088] In certain preferred embodiments, the polymerase used in probeelongation is a DNA polymerase that lacks 3′ to 5′ exonuclease activity.Examples of such polymerases include T7 DNA polymerase, T4 DNApolymerase, ThermoSequenase and Taq polymerase. When the target nucleicacid sequence is RNA, reverse transcriptase may be used. In addition topolymerase, nucleoside triphosphates are added, preferably all fourbases. For example dNTPs, or analogues, may be added. In certain otherembodiments, ddNTPs may be added. Labeled nucleotide analogues, such asCye3-dUTP may also be used to facilitate detection.

[0089] Prior art methods for detecting successful elongation have beendescribed which use labeled deoxy nucleoside triphosphates (dNTPs) ordideoxy nucleoside triphosphates (ddNTPs). This invention disclosesnovel methods of providing optical signatures for detecting successfulelongation, thus eliminating the need for labeled dNTPs or ddNTPs. Thisis advantageous because currently available polymerases are lessefficient in accommodating labeled dNTPs or ddNTPs.

[0090] This invention provides methods and compositions for accuratepolymorphism analysis of highly polymorphic target regions. As usedherein, highly polymorphic sequences are those containing, within aportion of the sequence contacted by the probe, not only the designatedor interrogated polymorphic site, but also non-designated polymorphicsites which represent a potential source of error in the analysis.Analogous considerations pertain to designs, compositions and methods ofmultiplexing PCR reactions. In a preferred embodiment, covering sets ofPCR probes composed of priming and annealing subsequences are displayedon encoded microparticles to produce bead-displayed amplicons by probeelongation. Assemblies of beads may be formed on planar substrates,prior to or subsequent to amplification to facilitate decoding andimaging of probes.

[0091] In one embodiment, this invention provides probes that aredesigned to contain a 3′ terminal “priming” subsequence, also referredto herein as a Terminal Elongation Initiation (TEI) region, and anannealing subsequence, also referred to herein as a Duplex Anchoring(DA) region. The TEI region typically comprises the three or four 3′terminal positions of a probe sequence. The TEI region is designed toalign with a portion of the target sequence at a designated polymorphicsite so as to initiate the polymerase-catalyzed elongation of the probe.Probe elongation indicates a perfect match in composition of the entireTEI region and the corresponding portion of the target sequence. The DAregion, comprising remaining positions within the probe sequence, ispreferably designed to align with a portion of the target sequence in aregion located close (within 3-5 bases) to the designated polymorphism.The duplex anchoring region is designed to ensure specific and strongannealing, and is not designed for polymorphism analysis. As describedherein, the DA and TEI regions may be located immediately adjacent toone another within the probe or may be linked by a molecular tether. Thelatter approach permits flexibility in the placement of DA region so asto avoid non-designated polymorphisms located immediately adjacent tothe designated site. The composition and length of the DA region arechosen to facilitate the formation of a stable sequence-specifichybridization complex (“duplex”), while accommodating (i.e., taking intoaccount) the presence of one or more non-designated polymorphismslocated in that region of the target. The length of the annealingsubsequence is chosen to minimize cross-hybridization by minimizingsequence homologies between probe and non-selected subsequences of thetarget. The length of the annealing subsequence generally exceeds thatof the priming subsequence so that failure to form a duplex generallyimplies failure to produce an elongation product.

[0092] The elongation reaction provides high specificity in detectingpolymorphisms located within the TEI region. For non-designatedpolymorphisms in the DA region, the elongation reaction will proceed ata level either comparable to, or lower than that of the perfect matchunder certain conditions. This is referred to as the tolerance effect ofthe elongation reaction. Tolerance is utilized in the design of probesto analyze designated and non-designated polymorphisms as described inexamples herein.

[0093] The density of polymorphic sites in the highly polymorphic lociconsidered in certain embodiments of this invention makes it likely thatprobes directed to designated polymorphic sites will overlap adjacentpolymorphic sites, when annealing to a target subsequence proximal tothe designated polymorphic site. That is, an oligonucleotide probedesigned to interrogate the configuration of the target at a selecteddesignated polymorphic site, and constructed with sufficient length toensure specificity and thermal stability in annealing to the correcttarget subsequence will align with nearby polymorphic sites. Theseinterfering polymorphic sites may include non-designated sites in thetarget sequence as well as designated but not selected polymorphic sites

[0094] Specifically, non-designated polymorphisms as contemplated in thepresent invention may interfere with duplex formation, therebyinterfering with or completely inhibiting probe elongation. In oneembodiment, the present invention provides designs of covering probesets to accommodate such non-designated polymorphisms. A covering probeset contains probes for concurrently interrogating a given multiplicityof designated polymorphic sites within a nucleic acid sequence. Acovering probe set comprises, for each site, at least one probe capableof annealing to the target so as to permit, on the basis of a subsequentelongation reaction, assignment of one of two possible values to thatsite: “matched” (elongation) or “unmatched”, (no elongation).

[0095] The covering probe set associated with each designated site maycontain two or more probes differing in one or more positions, alsoreferred to herein as a degenerate set. In certain embodiments, theprobe sequence may contain universal nucleotides capable of forming abase-pair with any of the nucleotides encountered in DNA. In certainembodiments, probes may be attached to encoded microparticles, andspecifically, two or more of the probes in a covering set or degenerateset may be attached to the same type of microparticle. The process ofattaching two or more probes to a microparticle or bead is referred toas “probe pooling”.

[0096] The design of covering probe sets is described herein inconnection with elongation-mediated multiplexed analysis ofpolymorphisms in two representative areas of genetic analysis: (1): thescoring of multiple uncorrelated designated polymorphisms and mutations,as in the case of mutation analysis for CF and Ashkenazi Jewish (AJ)disease carrier screening, and (2) the scoring of a correlated set ofpolymorphisms as in the case of HLA molecular typing. In the firstinstance, the covering set for the entire multiplicity of mutationscontains multiple subsets, each subset being associated with onedesignated site. In such a case, two or more probes are provided toascertain heterozygosity. For the purpose of general SNP identificationand confirmatory sequencing, degenerate probe sets can be provided tocontain up to four labeled (e.g., bead-displayed) probes per polymorphicsite. In the second instance, the covering set contains subsetsconstructed to minimize the number of probes in the set, as elaboratedherein. The set of designated probes is designed to identifyallele-specific sequence configurations on the basis of the elongationpattern.

[0097] While this method of accommodating or identifying non-designatedpolymorphic sites is especially useful in connection with themultiplexed elongation of sequence specific probes, it also may be usedin conjunction with single base extension of probes, also known asmini-sequencing (see e.g., Pastinen, et al. Genome Res. 7: 606-614(1997), incorporated herein by reference).

[0098] The elongation-mediated method of analysis of the presentinvention, unlike the single-base probe extension method, may be used todetect not only SNPs, but also to detect other types of polymorphismssuch as multiple (e.g., double, triple, etc.) nucleotide polymorphisms,as well as insertions and deletions commonly observed in the typing ofhighly polymorphic genetic loci such as HLA. In these complex systems,sequence-specific probe elongation in accordance with the methods ofthis invention, simplifies the detection step because two or more probesare provided for each polymorphic target location of interest and thedetection step is performed only to determine which of the two or moreprobes was elongated, rather than to distinguish between two extendedprobes, as in the case of single-base probe extension Thus, although themethods of this invention accommodate the use of multiple fluorophore orchromophore labels in the detection step, a single universal labelgenerally will suffice for the sequence specific probe elongation. Thisis in contrast to single-base extension methods whose application in amultiplexed format requires at least two fluorophore or chromophorelabels.

[0099] DNA methylation:

[0100] In certain embodiments, methods and compositions for determiningthe methylation status of DNA are provided. Cytosine methylation haslong been recognized as an important factor in the silencing of genes inmammalian cells. Cytosine methylation at single CpG dinucleotides withinthe recognition sites of a number of transcription factors is enough toblock binding and related to several diseases. eMAP can be used todetermine the methylation status of genomic DNA for diagnostic and otherpurposes. The DNA is modified by sodium bisulfite treatment convertingunmethylated Cytosines to Uracil. Following removal of bisulfite andcompletion of the chemical conversion, this modified DNA is used as atemplate for PCR. A pair of probes is designed, one specific for DNAthat was originally methylated for the gene of interest, and onespecific for unmethylated DNA. eMAP is performed with DNA polymerase andone labeled dNTP and unlabeled mixture of 3 dNTPs or ddNTPs. Theelongated product on the specific bead surface can indicate themethylation status.

[0101] Selective Sequencing:

[0102] In certain other embodiments of this invention, selectivesequencing (also referred to as “sequencing”) is used for concurrentinterrogation of an entire set of designated polymorphisms within anucleic acid sequence in order to determine the composition at each suchsite. Selective sequencing can be used to provide the requisiteinformation to select, from the set of possible configurations for thesequence of interest, the actual configuration in a given specificsample or to narrow the set of possible sequences in that sample. Inselective sequencing, the length of probes used in an extension reactiondetermine the length of the sequences that can be determined. For longerDNA sequences, staggered probe designs can be used to link the sequencestogether. Thus, known sequence combinations can be confirmed, whileunknown sequence combinations can be identified as new alleles.

[0103] Cystic Fibrosis Carrier Screening—

[0104] One practical application of this invention involves the analysisof a set of designated mutations within the context of a large set ofnon-designated mutations and polymorphisms in the Cystic FibrosisTransmembrane Conductance (CFTR) gene. Each of the designated mutationsin the set is associated with the disease and must be independentlyscored. In the simplest case of a point mutation, two encoded probes areprovided to ensure alignment of their respective 3′ termini with thedesignated site, with one probe anticipating the wild-type, and theother anticipating the altered (“mutated”) target sequence.

[0105] However, to ensure elongation regardless of the specific targetsequence configuration encountered near the designated site, additionalprobes are provided to match any of the possible or likelyconfigurations, as described in several Example herein. In a preferredembodiment, the covering probe set is constructed to contain probesdisplaying TEI sequences corresponding to all known or likely variationsof the corresponding target subsequence. This ensures elongation in thepresence of otherwise elongation-inhibiting non-designated polymorphismslocated within a range of proximity of the designated site.

[0106] In certain embodiments, the identification of the specific targetconfiguration encountered in the non-designated sites is not necessaryso long as one of the sequences provided in the covering probe setmatches the target sequence sufficiently closely to ensureelongation,and thus matches the target sequence exactly within the TEIregion. In this case, all or some of the covering probes sharing thesame 3′ terminus may be assigned the same code In a preferredembodiment, such probes may be associated with the same solid support(“probe pooling”). Probe pooling reduces the number of distinguishablesolid supports required to represent the requisite number of TEIsequences. In one particularly preferred embodiment, solid supports areprovided in the form of a set or array of distinguishable microparticleswhich may be decoded in-situ. Inclusion of additional probes in thecovering probe set to identify additional polymorphisms in the targetregion is a useful method to elucidate haplotypes for variouspopulations.

[0107] HLA—

[0108] Another application of this invention involves the geneticanalysis of the Human Leukocyte Antigen (HLA) complex, allowing theidentification of one or more alleles within regions of HLA encodingclass I HLA antigens (preferably HLA-A, HLA-B, HLA-C or any combinationthereof) and class II HLA antigens (preferably including HLA-DR, HLA-DQ,HLA-DP or any combination thereof). Class I and II gene loci also may beanalyzed simultaneously.

[0109] In contrast to the independent scoring of multiple uncorrelateddesignated mutations, identification of alleles (or groups of alleles)relies on the scoring of an entire set of elongation reactions. Each ofthese reactions involves one or more probes directed to a member of aselected set of designated polymorphic sites. The set of theseelongation reactions produces a characteristic elongation signalpattern. In a preferred embodiment, a binary pattern is produced,assigning a value of “1” to matching (and hence elongated) probes, and avalue of “0” to non-elongated probes. The binary pattern (“string”) ofgiven length uniquely identifies an allele or a group of alleles.

[0110] The total number of probes required for HLA typing depends on thedesired resolution. The term “resolution” is used here to indicate thedegree of allelic discrimination. Preferably, the method of thisinvention allows typing of an HLA allele that is sufficient todistinguish different antigen groups. For example, A*01 and A*03 aredifferent antigen groups that have to be distinguished in clinicalapplications. The National Marrow Donor Program (NMDP) recommended apanel for molecular typing of the donors. The low-to-medium resolutionrequired by the NMDP panel means that different antigen groups should bedistinguished at all times. Further, at least some of the alleles withinone group should be distinguished, though not necessarily all alleles.In certain embodiments, the present invention allows typing of the HLAallele to a low to medium resolution, as defined by the NMDP standard(www.NMDPresearch.org), incorporated herein by reference.

[0111] With such resolution, A*01, A*03 etc., will always be identified.A*0101 and A*0102 may not be necessarily distinguishable. For the SSOmethod, the current NMDP panel contains 30 probes for HLA-A; 48 forHLA-B and 31 for HLA-DR-B. High resolution HLA typing refers to thesituation when most of the alleles will be identified within each group.In this case, A*0101 and A*0102 will be distinguished. To reach suchresolution, approximately 500 to 1000 probes will be required for bothclass I and class II typing. In certain embodiments, the method of thepresent invention provides high resolution HLA typing, at least to thedegree described in Cao, et al., Rev. Immunogentics, 1: 177-208 (1999),incorporated herein by reference.

[0112] This invention also provides strategies for designating sites andfor designing probe sets for such designated sites in order to produceunique allele assignments based on the elongation reaction signalpatterns. The design of covering probes explicitly takes into accountthe distinct respective functions of TEI and DA regions of each probe.

[0113] A covering set of probes associated with a given designated siteis constructed to contain subsets. Each subset in turn contains probesdisplaying identical TEI regions. A mismatch in a single position withinthe TEI region, or a mismatch in three or more positions within the DAregion precludes elongation. Accordingly, the elongation of two probesdisplaying such differences in composition generally will producedistinct elongation patterns. All such probes can be multiplexed in aparallel elongation reaction as long as they are individually encoded.In a preferred embodiment, encoding is accomplished by attaching probesto color-encoded beads.

[0114] Probes displaying identical TEI subsequences and displaying DAsubsequences differing in not more than two positions generally willproduce elongation reactions at a yield (and hence signal intensity)either comparable to, or lower than that of a perfect match. In thefirst case which indicates tolerance of the mismatch, the set of allelesmatched by the probe in question will be expanded to include allelesthat display the tolerated mismatched sequence configurations within theDA region. In the second case, indicating only partial tolerance, threeapproaches are described herein to further elucidate the allele matchingpattern. In the first approach, probes displaying one or two nucleotidepolymorphisms in their respective DA regions are included in thecovering set. Information regarding the target sequence is obtained byquantitatively comparing the signal intensities produced by thedifferent probes within the covering set. In the second approach, probescomprising separate TEI and DA regions joined by a tether are used toplace the DA region farther away from the TEI region in order to avoidtarget polymorphisms. In the third approach, probes are optionallypooled in such cases offering only a modest expansion of the set ofmatched alleles.

[0115] In certain embodiments of this invention probes preferably aredesigned to be complementary to certain target sequences that are knownto correlate with allele combinations within the HLA gene locus. Knownpolymorphisms are those that have appeared in the literature or areavailable from a searchable database of sequences (e.g.,www.NMDProcessing.org). In certain embodiments, the HLA gene of interestbelongs to HLA class I group, (e.g., HLA-A, HLA-B or HLA-C orcombination thereof). In certain other embodiments, the HLA gene ofinterest belongs to the HLA class II group, (e.g., DR, DQ, DP orcombination thereof). The HLA class I and class II loci may be examinedin combination and by way of concurrent interrogation.

[0116] Probes previously employed in the SSP/gel method also may be usedin this invention. Preferably, the probes set forth in Bunce et al.,Tissue Antigen, 46: 355-367 (1995) and/or Bunce et al., Tissue Antigen,45:81-90 (1995), (each of which are hereby incorporated by reference)are used in preparing the probes for this invention. The probe sequencesor HLA sequence information provided in WO 00/65088; EuropeanApplication No. 98111696.5; WO 00/70006; and Erlich et al., Immunity,14: 347-356 (2001), (each of which are hereby incorporated by reference)may be used in designing the probes for this invention.

[0117] The complexity of an encoded bead array is readily adjusted toaccommodate the requisite typing resolution. For example, when 32 typesof beads are used for each of four distinct subarrays, a total of 128probes will be available to attain a medium level of resolution for HLAclass I and class II typing in a multiplexed elongation reaction.Analogously, with 128 types of beads and four subarrays, or 64 types ofbeads and 8 subarrays, a total of 512 probes will be available to attaina high resolution of HLA class I and class II typing in a multiplexedelongation reaction.

[0118] The encoded bead array format is compatible with high throughputanalysis. For example, certain embodiments of this invention provide acarrier that accommodates multiple samples in a format that iscompatible with the dimensions of 96-well microplates, so that sampledistribution may be handled by a standard robotic fluid handlingapparatus. This format can accommodate multiple encoded bead arraysmounted on chips and permits the simultaneous completion of multipletyping reactions for each of multiple patient samples on a singlemulti-chip carrier in a 96-well carrier testing 128 types per patient,more than 10,000 genotypes can be determined at a rate of throughputthat is not attainable by current SSP or SSO methodology.

[0119] In certain embodiments of this invention, the elongation reactioncan be combined with a subsequent hybridization reaction to correlatesubsequences on the same DNA target strand, a capability referred toherein as “phasing”. Phasing resolves ambiguities in allele assignmentarising from the possibility that a given elongation pattern isgenerated by different combinations of alleles. Similarly, phasing isuseful in the context of haplotying to assign polymorphisms to the sameDNA strand or chromosome.

[0120] In certain embodiments of this invention, the annealing andelongation steps of the elongation reaction can be combined as aone-step reaction. Furthermore, means to create continuous or discretetemperature variations can be incorporated into the system toaccommodate multiple optimal conditions for probes with differentmelting temperatures in a multiplexed reaction.

[0121] In certain embodiments of this invention, encoded bead arrays areformed on solid substrates. These solid substrates may comprise anysuitable solid material, such as glass or semiconductor, that hassufficient mechanical strength and can be subjected to fabricationsteps, if desired. In some embodiments, the solid substrates are dividedinto discrete units known as “chips”. Chips comprising encoded beadarrays may be processed individually or in groups, if they are loadedinto a multichip carrier. For example, standard methods of temperaturecontrol are readily applied to set the operating temperature of, or toapply a preprogramed sequence of temperature changes to, single chips orto multichip carriers. Further, chips may be analyzed with the directimaging capability of Random Encoded Array Detection (“READ”), asdisclosed in PCT/US01/20179, the contents of which are incorporatedherein by reference. Using READ, the multiplexed analysis of entirearrays of encoded beads on chips is possible. Furthermore, in the READformat, the application of preprogrammed temperature cycles providesreal-time on-chip amplification of elongation products. Given genomic,mitochondrial or other DNA, linear on-chip amplification may obviate theneed for pre-assay DNA amplification such as PCR, thereby dramaticallyshortening the time required to complete the entire typing assay.Time-sensitive applications such as cadaver typing are thereforepossible. More importantly, this approach eliminates the complexities ofPCR multiplexing, which is a limiting step in many genetic screening andpolymorphism analyses. In a preferred embodiment, a fluidic cartridgeprovides for sample and reagent injection as well as temperaturecontrol.

[0122] In one embodiment, the invention provides a method forpolymorphism analysis in which each target nucleic acid sequence is usedas a template in multiple elongation reactions by applying one or more“annealing-extending-detecting-denaturing” temperature cycles. Thismethod achieves linear amplification with in-situ detection of theelongation products. This additional capability obviates the need for afirst step of sequence-specific amplification of a polynucleotide sampleIntegration of assay procedure and signal amplification by way ofcycling not only simplifies and accelerates the completion of geneticanalysis, but also eliminates the need to develop, test and implementmultiplexed PCR procedures. The methods of this invention also provide ahigh-throughput format for the simultaneous genetic analysis of multiplepatient samples.

[0123] Several embodiments of this invention are provided for themultiplexed elongation of sequence-specific probes to permitsimultaneous evaluation of a number of different targets. In certainembodiments, oligonucleotide probes are immobilized on a solid supportto create dense patterns of probes on a single surface, e.g., silicon orglass surface. In certain embodiments, presynthesized oligonucleotideprobes are immobilized on a solid support, examples of which includesilicon, chemically modified silicon, glass, chemically modified glassor plastic. These solid supports may be in the form of microscopicbeads. The resolution of the oligonucleotide array is determined by bothspatial resolution of the delivery system and the physical spacerequirements of the delivered nucleotide solution volume. [See Guo, etal., Nucleic Acids Res. 22: 5456-5465 (1994); Fahy, et al., Nucleic AcidRes. 21: 1819-1826 (1993); Wolf, et al., Nuc. Acids Res. 15: 2911-2926(1987); and Ghosh, et al., Nuc. Acids Res. 15: 5353-5372 (1987).]

[0124] This invention provides methods for multiplexed assays. Incertain embodiments, sets of elongation probes are immobilized on asolid phase in a way that preserves their identity, e.g., by spatiallyseparating different probes and/or by chemically encoding the probeidentities. One or more solution-borne targets are then allowed tocontact a multiplicity of immobilized probes in the annealing andelongation reactions. This spatial separation of probes from one anotherby immobilization reduces ambiguities in identifying elongationproducts. Thus, this invention offers advantages over the existingPCR-SSP method, which is not adaptable to a high throughput formatbecause of (i) its requirement for two probes for each PCRamplification; (ii) the competition between overlapping probes for thehighly polymorphic genes, such as HLA, in a multiplexed homogeneousreaction; and (iii) the difficulty in distinguishing between specificproducts in such a multiplexed reaction.

[0125] In a preferred embodiment, probes are attached, via theirrespective 5′ termini, to encoded microparticles (“beads”) having achemically or physically distinguishable characteristic that uniquelyidentifies the attached probe. Probes capture target sequences ofinterest contained in a solution that contacts the beads. Elongation ofthe probe displayed on a particular bead produces an opticallydetectable signature or a chemical signature that may be converted intoan optically detectable signature. In a multiplexed elongation reaction,the optical signature of each participating bead uniquely corresponds tothe probe displayed on that bead. Subsequent to the probe elongationstep, one may determine the identity of the probes by way of particleidentification and detection, e.g., by flow cytometry.

[0126] In certain embodiments, beads may be arranged in a planar arrayon a substrate before the elongation step. Beads also may be assembledon a planar substrate to facilitate imaging after the elongation step.The process and system described herein provide a high throughput assayformat permitting the instant imaging of an entire array of beads andthe simultaneous genetic analysis of multiple patient samples.

[0127] The array of beads may be a random encoded array, in which achemically or physically distinguishable characteristic of the beadswithin the array indicates the identity of oligonucleotide probesattached to the beads. The array may be formed according to the READformat The bead array may be prepared by employing separate batchprocesses to produce application-specific substrates (e.g., a chip atthe wafer scale). Beads that are encoded and attached to oligonucleotideprobes (e.g., at the scale of about 10⁸ beads/100 μl suspension) arecombined with a substrate (e.g., silicon chip) and assembled to formdense arrays on a designated area of the substrate. In certainembodiments, the bead array contains 4000 beads of 3.2 μm diameter andhas a dimension of 300 μm by 300 μm. With beads of different size, thedensity will vary. Multiple bead arrays also can be formedsimultaneously in discrete fluid compartments maintained on the samechip. Such methods are disclosed in U.S. application Ser. No.10/192,351, filed Jul. 9, 2002, which is incorporated herein byreference in its entirety.

[0128] Bead arrays may be formed by the methods collectively referred toas “LEAPS”, as described in U.S. Pat. No. 6,251,691 and PCTInternational Application No. PCT/US00/25466),both of which areincorporated herein by reference.

[0129] The substrate (e.g., a chip) used in this invention may be in theform of a planar electrode patterned in accordance with the interfacialpatterning methods of LEAPS. For example, the substrate may be patternedwith oxide or other dielectric materials to create a desiredconfiguration of impedance gradients in the presence of an applied ACelectric field. Patterns may be designed so as to produce a desiredconfiguration of AC field-induced fluid flow and corresponding particletransport. Substrates may be patterned on a wafer scale by usingsemiconductor processing technology. In addition, substrates may becompartmentalized by depositing a thin film of a UV-patternable,optically transparent polymer to affix to the substrate a desired layoutof fluidic conduits and compartments. These conduits and compartmentsconfine fluid in one or several discrete compartments, therebyaccommodating multiple samples on a given substrate.

[0130] Bead arrays may be prepared using LEAPS by providing a firstplanar electrode that is in substantially parallel to a second planarelectrode (“sandwich” configuration) with the two electrodes beingseparated by a gap and containing a polarizable liquid medium, such asan electrolyte solution. The surface or the interior of the secondplanar electrode may be patterned with the interfacial patterningmethod. The beads are introduced into the gap. When an AC voltage isapplied to the gap, the beads form a random encoded array on the secondelectrode (e.g., a “chip”).

[0131] In another embodiment of LEAPS, an array of beads may be formedon a light-sensitive electrode (e.g., a “chip”). Preferably, thesandwich configuration described above is also used with a planar lightsensitive electrode and another planar electrode. Once again, the twoelectrodes are separated by the a gap and contain an electrolytesolution. The functionalized and encoded beads are introduced into thegap. Upon application of an AC voltage in combination with light, thebeads form an array on the light-sensitive electrode.

[0132] In certain embodiments of the present invention, beads may beassociated with a chemically or physically distinguishablecharacteristic. This may be provided, for example, by staining beadswith sets of optically distinguishable tags, such as those containingone or more fluorophore or chromophore dyes spectrally distinguishableby excitation wavelength, emission wavelength, excited-state lifetime oremission intensity. The optically distinguishable tags may be used tostain beads in specified ratios, as disclosed, for example, in Fulwyler,U.S. Pat. No. 4,717,655 (Jan. 5, 1988). Staining may also beaccomplished by swelling of particles in accordance with methods knownto those skilled in the art, (Molday, Dreyer, Rembaum & Yen, J. Mol Biol64, 75-88 (1975); L. Bangs, “Uniform latex Particles, SeragenDiagnostics, 1984). For example, up to twelve types of beads wereencoded by swelling and bulk staining with two colors, each individuallyin four intensity levels, and mixed in four nominal molar ratios.Alternatively, the methods of combinatorial color encoding described inInternational Application No. PCT/US 98/10719 (incorporated by referencein its entirety) can be used to endow the bead arrays with opticallydistinguishable tags. In addition to chemical encoding, beads may alsobe rendered magnetic by the processes described in PCT/US0/20179.

[0133] In addition to chemical encoding with dyes, beads having certainoligonucleotide primers may be spatially separated (“spatial encoding”),such that the location of the beads provides information as to theidentity of the beads. Spatial encoding, for example, can beaccomplished within a single fluid phase in the course of array assemblyby using Light-controlled Electro kinetic Assembly of Particles nearSurfaces (LEAPS). LEAPS can be used to assemble planar bead arrays inany desired configuration in response to alternating electric fieldsand/or in accordance with patterns of light projected onto thesubstrate.

[0134] LEAPS can be used to create lateral gradients in the impedance atthe interface between a silicon chip and a solution to modulate theelectrohydrodynamic forces that mediate array assembly. Electricalrequirements are modest: low AC voltages of typically less than 10V_(pp)are applied across a fluid gap between two planar electrodes that istypically 100 μm. This assembly process is rapid and it is opticallyprogrammable: arrays containing thousands of beads are formed withinseconds under an applied electric field. The formation of multiplesubarrays can also occur in multiple fluid phases maintained on acompartmentalized chip surface.

[0135] Subsequent to the formation of an array, the array may beimmobilized. For example, the bead arrays may be immobilized, forexample, by application of a DC voltage to produce random encodedarrays. The DC voltage, set to typically 5-7 V (for beads in the rangeof 2-6 μm and for a gap size of 100-150 μm) and applied for <30 s in“reverse bias” configuration so that an n-doped silicon substrate wouldform the anode, causes the array to be compressed to an extentfacilitating contact between adjacent beads within the array andsimultaneously causes beads to be moved toward the region of highelectric field in immediate proximity of the electrode surface. Once insufficiently close proximity, beads are anchored by van der Waals forcesmediating physical adsorption. This adsorption process is facilitated byproviding on the bead surface a population of “tethers” extending fromthe bead surface; polylysine and streptavidin have been used for thispurpose.

[0136] In certain embodiments, the particle arrays may be immobilized bychemical means, e.g, by forming a composite gel-particle film. In oneexemplary method for forming such gel-composite particle films, asuspension of microparticles is provided which also contains monomer,crosslinker and initiator for in-situ gel formation. The particles areassembled into a planar assembly on a substrate by using LEAPS. ACvoltages of 1-20 V_(p-p) in a frequency range from 100's of hertz toseveral kilohertz are applied between the electrodes across the fluidgap. In the presence of the applied AC voltage, polymerization of thefluid phase is triggered after array assembly by thermally heating thecell to ˜40-45° C. using an infra-red (IR) lamp or photoinitiating thereaction using a mercury lamp source. The resultant gel effectivelyentraps the particle array. Gels may be composed of a mixture ofacrylamide and bisacrylamide of varying monomer concentrations from 20%to 5% (acrylamide:bisacrylamide=37.5:1, molar ratio), but any other lowviscosity water soluble monomer or monomer mixture may be used as well.Chemically immobilized functionalized microparticle arrays prepared bythis process may be used for a variety of bioassays, e.g., ligandreceptor binding assays.

[0137] In one example, thermal hydrogels are formed usingazodiisobutyramidine dihydrochloride as a thermal initiator at a lowconcentration to ensure that the overall ionic strength of thepolymerization mixture falls in the range of ˜0.1 mM to 1.0 mM. Theinitiator used for the UV polymerization is Irgacure 2959®(2-Hydroxy-4′-hydroxyethoxy-2-methylpropiophenone, Ciba Geigy,Tarrytown, N.Y.). The initiator is added to the monomer to give a 1.5%by weight solution.

[0138] In certain embodiments, the particle arrays may be immobilized bymechanical means. For example, an array of microwells may be produced bystandard semiconductor processing methods in the low impedance regionsof a silicon substrate. Particle arrays may be formed using suchstructures. In certain embodiments LEAPS mediated hydrodynamic andponderomotive forces are utilized to transport and to accumulateparticles on the hole arrays. The AC field is then switched off andparticles are trapped into microwells and thus mechanically confined.Excess beads are removed leaving behind a spatially ordered random beadarray on the substrate surface.

[0139] Substrates (e.g., chips) can be placed in one or more enclosedcompartments that permit samples and reagents to be transported in andout of the compartments through fluidic interconnection. Reactions canalso be performed in an open compartment format such as a microtiterplate. Reagents may be pipetted on top of the chip by robotic liquidhandling equipment, and multiple samples may be processedsimultaneously. Such a format accommodates standard sample processingand liquid handling for the existing microtiter plate format andintegrates sample processing and array detection.

[0140] In certain embodiments of this invention, encoded beads areassembled on the substrate surface, but not in an array. For example, byspotting bead suspensions into multiple regions of the substrate andallowing beads to settle under gravity, assemblies of beads can beformed on the substrate. In contrast to the bead arrays formed by LEAPS,these assemblies generally assume disordered configurations oflow-density or non-planar configurations involving stacking or clumpingof beads, thereby preventing imaging of affected beads. However, thecombination of spatial and color encoding attained by spotting mixturesof chemically encoded beads into a multiplicity of discrete positions onthe substrate still allows multiplexing.

[0141] In certain embodiments, a comparison of an image of an arrayafter the assay with a decoded image of the array can be used to revealchemically or physically distinguishable characteristics, as well as theelongation of probes. This comparison can be achieved by using, forexample, an optical microscope with an imaging detector and computerizedimage capture and analysis equipment. The assay image of the array istaken to detect the optical signature that indicates the probeelongation. The decoded image is taken to determine the chemicallyand/or physically distinguishable characteristics that uniquely identifythe probe displayed on the bead surface. In this way, the identity ofthe probe on each particle in the array may be identified by adistinguishable characteristic.

[0142] Image analysis algorithms may be used in analyzing the dataobtained from the decoding and the assay images. These algorithms may beused to obtain quantitative data for each bead within an array. Theanalysis software automatically locates bead centers using abright-field image of the array as a template, groups beads according totype, assigns quantitative intensities to individual beads, rejects“blemishes” such as those produced by “matrix” materials of irregularshape in serum samples, analyzes background intensity statistics andevaluates the background-corrected mean intensities for all bead typesalong with the corresponding variances. Examples of such algorithms areset forth in PCT/US01/20179.

[0143] Probe elongation may be indicated by a change in the opticalsignature, or a change in chemical signature which may be converted to achange in optical signature, originating from the beads displayingelongated probes, for example. Direct and indirect labeling methods wellknown in the art are available for this purpose. Direct labeling refersto a change in optical signature resulting from the elongation; indirectlabeling refers to a change introduced by elongation which requires oneor more additional steps to produce a detectable optical signature. Incertain embodiments, fluorophore or chromophore dyes may be attached toone of the nucleotides added as an ingredient of probe elongation, suchthat probe elongation changes the optical signature of beads bychanging, for example, fluorescence intensities or by providing otherchanges in the optical signatures of beads displaying elongationproducts.

EXAMPLES

[0144] The present invention will be better understood from the Exampleswhich follow. It should be understood that these examples are forillustrative purposes and are not to be construed as limiting thisinvention in any manner.

Example 1 Staggered Probe Design for Multiplexed SSP Analysis

[0145] Probes for each polymorphism are immobilized on a solid phasecarrier to provide a format in which multiple concurrent annealing andextension reactions can proceed with minimal mutual interference.Specifically, this method provides a design which accommodatesoverlapping probes, as illustrated in FIG. 1. In this example, weconsider three alleles: allele A, allele B and allele C. Probes 1 and 2detect SNPs that are aligned with their respective 3′ termini whileprobes 3 and 4 detect two-nucleotide polymorphisms that are aligned withtheir respective 3′ termini. The polymorphic sites targeted by probes 1and 2 are located five nucleotides upstream of those targeted by probes3 and 4. This design permits each probe to bind its corresponding targetand permits elongation to proceed when there is a perfect match at thedesignated polymorphic site. Thus, probes 1 and 3 match allele A, probe2 and possibly probe 3 match allele B, and probes 1 and 4 match allele C

Example 2 Probe Design for HLA Typing

[0146] To design probes for the analysis of the polymorphic regionranging from base 106 to base 125 of the DRB gene, twenty-two differenttypes of sequences for the 20 base long fragment were located in the DRBdatabase. These are listed in the table below: 7 DRB1*0101TTCTTGTGGCAGCTTAAGTT 104 DRB1*03011 TTCTTGGAGTACTCTACGTC 26 DRB1*04011TTCTTGGAGCAGGTTAAACA 1 DRB1*0434 TTCTTGGAGCAGGTTAAACC 3 DRB1*07011TTCCTGTGGCAGGGTAAGTA 1 DRB1*07012 TTCCTGTGGCAGGGTAAATA 28 DRB1*0801TTCTTGGAGTACTCTACGGG 1 DRB1*0814 TTCTTGGAGTACTCTAGGGG 1 DRB1*0820TTCTTGGAGTACTCTACGGC 1 DRB1*0821 TTCTTGGAGTACTCTATGGG 1 DRB1*09012TTCTTGAAGCAGGATAAGTT 2 DRB1*10011 TTCTTGGAGGAGGTTAAGTT 1 DRB1*1122TTCTTGGAGCAGGCTACACA 1 DRB1*1130 TTCTTGGAGTTCCTTAAGTC 18 DRB1*15011TTCCTGTGGCAGCCTAAGAG 9 DRB3*01011 TTCTTGGAGCTGCGTAAGTC 1 DRB3*0102TTCTTGGAGCTGTGTAAGTC 1 DRB3*0104 TTCTCGGAGCTGCGTAAGTC 16 DRB3*0201TTCTTGGAGCTGCTTAAGTC 1 DRB3*0212 TTCTTGCAGCTGCTTAAGTC 6 DRB4*01011TTCTTGGAGCAGGCTAAGTG 14 DRB5*01011 TTCTTGCAGCAGGATAAGTA

[0147] The first column contains the number of alleles sharing thesequence listed in third column, the second column contains one of theallele names. We selected the last three bases of the 20-base fragmentas the TEI region and sorted the set of sequences according to their TEIregion to obtain the following groups: 1 104 DRB1*03011TTCTTGGAGTACTCTACGTC e1 1 DRB1*1130 TTCTTGGAGTgCctTAaGTC 9 DRB3*01011TTCTTGGAGctgcgTAaGTC 1 DRB3*0102 TTCTTGGAGctgTgTAaGTC 1 DRB3*0104TTCTcGGAGctgcgTAaGTC 16 DRB3*0201 TTCTTGGAGctgctTAaGTC e2 1 DRB3*0212TTCTTGcAGctgctTAaGTC 2 7 DRB1*0101 TTCTTGTGGCAGCTTAAGTT 1 DRB1*09012TTCTTGaaGCAGgaTAAGTT 2 DRB1*10011 TTCTTGgaGGAGgTTAAGTT 3 26 DRB1*04011TTCTTGGAGCAGGTTAAACA 1 DRB1*1122 TTCTTGGAGCAGGcTAcACA 4 1 DRB1*0434TTCTTGGAGCAGGTTAAACC 5 3 DRB1*07011 TTCCTGTGGCAGGGTAAGTA 14 DRB5*01011TTCtTGcaGCAGGaTAAGTA 6 1 DRB1*07012 TTCCTGTGGCAGGGTAAATA 7 28 DRB1*0801TTCTTGGAGTACTCTACGGG e3 1 DRB1*0814 TTCTTGGAGTACTCTAgGGG 1 DRB1*0821TTCTTGGAGTACTCTAtGGG 8 1 DRB1*0820 TTCTTGGAGTACTCTACGGC 9 18 DRB1*15011TTCCTGTGGCAGCCTAAGAG 10 6 DRB4*01011 TTCTTGGAGCAGGCTAAGTG

[0148] For sequences in the same group, variations between the firstsequence of the group and the rest are indicated in lower case. Threeprobe sequences are used to illustrate the application of our probedesign rules. The first sequence in the first group is selected as probee1; the 6th sequence in the first group is selected as probe e2; and thefirst group in the 7th sequence is selected as probe e3.

[0149] Due to requirement for perfect complementarity of the target andthe probe's TEI region, sequences in group to group 10 do not produceelongation products for e1 and e2. Similarly, sequences in groups otherthan the 7th group do not produce elongation products for e3. Each groupis distinctive from the others with respect to elongation reactionpatterns.

[0150] For sequences in the same group, there are two types ofsituations. For example, e1 and e2 differ by one nucleotide in 6positions within the annealing region. Thus, targets matching e1 and e2will not produce elongation products for the other sequences, and e1 ande2 are also distinct probes.

[0151] Similarly, targets for the second to the 7th sequences in group 1will not produce elongation products for probe e1.

[0152] Except for the target matching e1, the remaining 5 sequences onlydiffer from e2 by one or two nucleotides as indicated below:1,2................M 16 DRB3*0201 TTCTTGGAGCTGCTTAAGTC e2 1 DRB1*1130TTCTTGGAGtTcCTTAAGTC a 9 DRB3*01011 TTCTTGGAGCTGCgTAAGTC b 1 DRB3*0102TTCTTGGAGCTGtgTAAGTC c 1 DRB3*0104 TTCTcGGAGCTGCgTAAGTC d 1 DRB3*0212TTCTTGcAGCTGCTTAAGTC e

[0153] These sequences are cross-reactive. When targets for sequences band e, which differ from e2 by one base at respective positions M−7 andM−14 anneal to probe e2, the non-designated poylmorphism(s) in theannealing region will be tolerated and the elongation reaction willproceed to substantially the same degree as for perfectly matchedsequences. When targets for sequences a, c, and d, which differ from e2by two nucleotides anneal to probe e2, the elongation reaction willexhibit only partial tolerance of the non-designated polymoprhism(s).One approach to improve on this situation is to provide separate probesfor a, c, and d, then quantitatively analyze the yield of elongationproducts by analyzing signals intensitities to identify the correctsequences. An alternative would be to bridge the non-designatedpolymorphisms in the annealing region altogether by adding a physicallinker (e.g., a tether) to the e2 probe to be able to separate annealingand TEI regions

[0154] For the sequences in the 7th group, the other two sequences willbe partially tolerated by the e3 probe. These three sequences may bepooled. The e2 probe will yield elongation products for 30 allelesinstead of 28 alleles.

Example 3 Utilizing Mismatch Tolerance to Modify Allele Binding Patterns

[0155] Probe DR-13e, GGACATCCTGGAAGACGA, was used to target the bases281-299 of the DRB gene. Thirty-four alleles, including alleleDRB1*0103, are perfectly matched to this sequence. Thus, in the bindingpattern, 13e is positive for theses 34 alleles (that is, 13e will yieldelongation products with these 34 alleles). Several additional allelesdisplay the same TEI region but display non-designated polymorphisms intheir respective annealing regions. For example, five alleles, such asDRB1*0415, contain T in instead of A in position 4 while four alleles,such as DRB1*1136,contain C in the that position. Due to mismatchtolerance in the annealing region, target sequences complementary tothese nine alleles will produce elongation reaction patterns similar tothat of the perfectly matched sequence. The result is shown in FIG. 2.TO-3 and TO-4 are completely complementary sequences to allele *0415 and*1136, respectively. DRB1*0103 GACATCCTGGAAGACGA 34 alleles DRB1*0415GACTTCCTGGAAGACGA  5 alleles DRB1*1136 GACCTCCTGGAAGACGA  4 alleles

Example 4 Design of Linker Structure in the Probes to BridgeNon-Designated Polymorphisms

[0156] As illustrated in FIG. 3, an anchor sequence is derived fromconserved sequence regions to ensure specific and strong annealing. Itis not designed for polymorphism detection. For that purpose, a shortersequence for polymorphism detection is attached to the anchoringsequence by way of a neutral chemical linker. The shorter length of thesequence designed for polymorphism detection will limit potentialinterference to non-designated polymorphisms in the immediate vicinityof the designated site and thus decreases the number of possiblesequence combinations required to accommodate such interferingpolymorphisms This approach avoids highly dense polymorphic sites incertain situations. For example, it would be possible to distinguishbetween the sequences listed in Example 3 using a probe which takes intoaccount the additional polymorphism(s). Illustrative designs of thelinker and the sequences are listed below: linker AGCCAGAAGGAC/Spacer18/spacer 18/GGAAGACGA 13-5 linker AGCCAGAAGGAC/Spacer 18/spacer18/AGACGA 13-8 linker AGCCAGAAGGAC/Spacer 18/spacer 18/CGA 13-11

Example 5 Phasing

[0157] The present invention also is useful in reducing ambiguities thatarise when two or more allele combinations can produce the same reactionpattern. In a simulated situation shown in FIGS. 4 and 5, allele A whichmatches—and hence produces an elongation product with—Probe 1 and Probe3, and allele B, which matches Probe 2 and Probe 4 when present in thesame multiplexed reaction, generate the same total reaction pattern asdoes the combination of allele C which matches Probe 1 and 2, and alleleD which matches Probe 3 and and Probe 4. Such ambiguity can be reducedor eliminiated by using the detection methods provided in this inventionto analyze the elongation product of Probe 1 by hybridization using alabeled detection probe that is designed to target the same polymorphicsite as Probe 3. If the result of the analysis is positive, only oneallele combination, namely combination 1, is possible because Probe 1and Probe 3 are associated with the same allele. The detection probe canbe labeled by using any of the methods disclosed in this invention ormethods known in the art. If this identification detection step isperformed together with the multiplexed elongation reaction detection,different labels are used for the elongation detection and probehybridization detection as shown in the FIG. 5.

[0158] In this method, the ambiguity is resolved by assigning two ormore polymorphisms to the same “phase” using elongation in conjunctionwith hybridization. Phasing is rapidly emerging as an important concernfor haplotype analysis in other genetic studies designed in the art.More probes can be included by reacting them with the targetsequentially, or they can be arranged in the same reaction withdifferent labels for detection.

[0159] The capability of combining probe elongation and hybridizationreactions is demonstrated in experiments using a sample sequence fromHLA-B exon 3. The result is shown in FIG. 6. A probe SB3P was elongatedin the reaction and the elongated product was detected using a labeledDNA probe. For the two samples presented in FIGS. 6A and 6B, SB 127r andSB3P, and SB285r and SB3P are in the same phase, respectively.

Example 6 Model HLA Typing Reaction Using Random Encoded Probe Arrays

[0160] To illustrate the discrimination of polymorphisms, a modelreaction was performed using a synthetic single strand as the target.Color encoded, tosyl-functionalized beads of 3.2 μm diameter were usedas solid phase carriers. A set of 32 distinguishable color codes wasgenerated by staining particles using standard methods known in the art(Bangs. L. B., “Uniform Latex Particles”, Seragen Diagnostics Inc.,p.40) and using different combinations of blue dye (absorption/emission419/466 nm) and green dye (absorption/emission 504/511). Stained beadswere functionalized with Neutravidin (Pierce, Rockford, Ill.), a biotinbinding protein, to mediate immobilization of biotinylated probes. In atypical small-scale coupling reaction, 200 μl of suspension containing1% beads were washed three times with 500 μl of 100 mM phosphatebuffer/pH 7.4 (buffer A) and resuspended in 500 μl of that buffer. Afterapplying 20 μl of 5 mg/ml neutravidin to the bead suspension, thereaction was sealed and allowed to proceed overnight at 37° C. Coupledbeads were then washed once with 500 μl of PBS/pH 7.4 with 10 mg/ml BSA(buffer B), resuspended in 500 μl of that buffer and reacted for 1 hourat 37° C. to block unreacted sites on bead surface. After blocking,beads were washed three times with buffer B and stored in 200 μl of thatbuffer.

[0161] In the model reaction system, two pairs of probes weresynthesized to contain SNPs at their respective 3′ termini. Therespective sequences were as follows: SSP13: AAGGACATCCTGGAAGACG; SSP24:AAGGACATCCTGGAAGACA; SSP16: ATAACCAGGAGGAGTTCC SSP36:ATAACCAGGAGGAGTTCG.

[0162] The probes were biotinylated at the 5′ end; a 15-carbontriethylene glycol linker was inserted between biotin and theoligonucleotide to minimize disruptive effects of the surfaceimmobilization on the subsequent reactions. For each probe, coupling toencoded beads was performed using 50 μl of bead suspension. Beads werewashed once with 500 μl of 20 mM Tris/pH 7.4, 0.5M NaCl (buffer C) andresuspended in 300 μl of that buffer. 2.5 μl of a 100 μM solution ofprobe were added to the bead suspension and allowed to react for 30 minat room temperature. Beads were then washed three times with 20 mMTris/pH7.4, 150 mM NaCl, 0.01% triton and stored in 20 mM Tris/pH 7.4,150 mM NaCl.

[0163] The following synthetic targets of 33 bases in length wereprovided: TA16: GTCGAAGCGCAGGAACTCCTCCTGGTTATGGAA TA36:GTCGAAGCGCACGAACTCCTCCTGGTTATAGAA TA13:GGCCCGCTCGTCTTCCAGGATGTCCTTCTGGCT TA24:GGCCCGCTTGTCTTCCAGGATGTCCTTCTGGCT

[0164] Targets were allowed to react with four probes (SSP13, SSP24,SSP16, SSP36) on the chip. An aliquot of 10 μl of a 100 nM solution ofthe target in annealing buffer of 0.2 M NaCl, 0.1% Triton X-100, 10 mMTris/pH 8.0, 0.1 mM EDTA was applied to the chip and allowed to reactfor 15 min at 30 ° C. The chip was then washed once with the same bufferand was then covered with an extension reaction mixture including: 100nM of TAMRA-ddCTP (absorption/emission: 550/580) (Perkin ElmerBioscience, Boston, Mass.), 10 μM dATP-dGTP-dTTP, ThermoSequenase(Amersham, Piscataway, N.J.) in the associated buffer supplied by themanufacturer. The reaction was allowed to proceed for 5 min at 60° C.,and the chip was then washed in H₂O. Decoding and assay images of thechip were acquired using a Nikon fluorescence E800 microscope with anautomated filter changer containing hydroxy coumarin, HQ narrow band GFPand HQ Cy3 filters for blue, green decoding images and for the assayimage, respectively. An Apogee CCD KX85 (Apogee Instruments, Auburn,Calif.) was used for image acquisition. In each reaction, only theperfectly matching target was extended producing, in the case of theSNPs tested here, discrimination between matching and non-matchingtargets in the range from 13-fold to 30-fold; this is illustrated inFIG. 7 for TA13.

Example 7 HLA-DR Typing of Patient Sample

[0165] A DNA sample extracted from a patient was processed using astandard PCR protocol. The following primers were used for general DRamplification: forward primer: GATCCTTCGTGTCCCCACAGCACG reverse primer:     GCCGCTGCACTGTGAAGCTCTC.

[0166] The PCR protocol was as follows: one cycle of 95° C. for 7 min,35 cycles of 95° C. for 30 sec, 60° C. for 30 sec and 72° C. for 1 minand one cycle of 72° C. for 7 min.

[0167] The PCR product, 287 bases in length and covering the DR locus,was denatured at 100° C. for 5 min, chilled on ice and mixed withannealing buffer as described in Example 6 for the model reaction. Analiquot of 10 ul was applied to each chip and reacted at 40° C. for 15min. The elongation reaction and subsequent image acquisition proceededas in the previous Example 6.

[0168] The multiplexed extension of sequence-specific probes using thePCR product produced from the patient sample produced results inaccordance with the probe design. Of the four probes tested in parallel(SSP13, SSP16, SSP24, SSP36), SSP13 was elongated while the SNP probeSSP24 only showed background binding as did the unrelated SSP16 andSSP36 probes. As illustrated in FIG. 8, the multiplexed elongation ofSSP significantly enhanced the discrimination between matching andnon-matching SNPs from approximately two-fold for an analysis based onthe hybridization of matching and non-matching sequence-specificoligonucleotide probes to at least 20-fold.

Example 8 Group-Specific Amplification

[0169] Primers for group-specific amplification (GSA) are mostfrequently used when multiplexed hybridization with SSOs yieldsambiguous assignments of heterozygous allele combinations. In such asituation, GSA primers are selected to amplify selected sets of specificalleles so as to remove ambiguities, a labor-intensive additional assaystep which delays the analysis. Using the methods of the presentinvention, preferably an embodiment of displaying probes on randomencoded bead arrays, GSA primers may be incorporated as probes into themultiplexed reaction thereby eliminating an entire second step ofanalysis.

Example 9

[0170] Analysis of HLA-DR, -A and -B Loci Using Cell Lines

[0171] Probes for the elongation-mediated multiplexed analysis ofHLA-DR, HLA-A and HLA-B were designed and tested using standard celllines. The probes were derived from SSP probes previously reported inthe literature (Bunce, M. et al, Tissue Antigens. 46:355-367 (1995),Krausa, P and Browning, M. J., Tissue Antigens. 47: 237-244 (1996),Bunce, M. et al, Tissue Antigens. 45:81-90 (1995)).

[0172] The probes used for DR were: SR2: ACGGAGCGGGTGCGGTTG SR3:GCTGTCGAAGCGCACGG SR11: CGCTGTCGAAGCGCACGTT SR19:GTTATGGAAGTATCTGTCCAGGT SR23: ACGTTTCTTGGAGCAGGTTAAAC SR32:CGTTTCCTGTGGCAGGGTAAGTATA SR33: TCGCTGTCGAAGCGCACGA SR36:CGTTTCTTGGAGTACTCTACGGG SR39: TCTGCAGTAGGTGTCCACCA SR45:CACGTTTCTTGGAGCTGCG SR46: GGAGTACCGGGCGGTGAG SR48:GTGTCTGCAGTAATTGTCCACCT SR52: CTGTTCCAGGACTCGGCGA SR57:CTCTCCACAACCCCGTAGTTGTA SR58: CGTTTCCTGTGGCAGCCTAAGA SR60:CACCGCGGCCCGCGC SR67: GCTGTCGAAGCGCAAGTC SR71: GCTGTCGAAGCGCACGTA NEGAAAAAAAAAAAAAAAAAA

[0173] Some of the probes have a SNP site at their respective 3′termini, for example: SR3 and SR33 (G and A, respectively); SR11, SR67and SR71 (T,C, and A, respectively). In addition, probes SR3 and 33 arestaggered at the 3′-end with respect to probes the group of SR11, 67 and71 by one base. SR3 GCTGTCGAAGCGCACGG SR33 TCGCTGTCGAAGCGCACGA SR11CGCTGTCGAAGCGCACGTT SR67 GCTGTCGAAGCGCAAGTC SR71 GCTGTCGAAGCGCACGTA

[0174] Reaction conditions were as described in Example 7 except thatthe annealing temperature was 55° C. instead of 40° C., and theextension temperature was 70° C. instead of 60° C. Double-stranded DNAwas used as in Example 7. Single-stranded DNA generated better resultsunder current conditions. Single-stranded DNA was generated byre-amplifying the initial PCR product in the same PCR program with onlyone of the probes. Results for two cell lines, W51 and SP0010, are shownin FIG. 9 and FIG. 10. NEG, a negative control, was coupled to aselected type of bead. Signal intensity for other probes minus NEG wasconsidered to be real signal for the probe and the values were plottedin the figures. The Y axis unit was the signal unit from the camera usedin the experiment. The distinction between the positive and negativeprobes was unambiguous for each sample. In particular, and in contrastto the situation typically encountered in SSO analysis, it was notnecessary to make comparisons to other samples to determine a reliablethreshold for each probe.

[0175] The probes used for HLA-A were: SAD CACTCCACGCACGTGCCA SAFGCGCAGGTCCTCGTTCAA SAQ CTCCAGGTAGGCTCTCAA SAR CTCCAGGTAGGCTCTCTG SAXGCCCGTCCACGCACCG SAZ GGTATCTGCGGAGCCCG SAAP CATCCAGGTAGGCTCTCAA SA8GCCGGAGTATTGGGACGA SA13 TGGATAGAGCAGGAGGGT SA16 GACCAGGAGACACGGAATA

[0176] Results for A locus exon 3, shown in FIG. 11 and FIG. 12, alsowere unambiguous. FIG. 12 also shows an example of the mismatchtolerance for a non-designated polymorphism. That is, while allele 0201,displaying C instead of A at position M−18, is not perfectly matched toprobe SAAP, the elongation reaction nonetheless proceeded because thepolymerase detected a perfect match for the designated polymorphism atthe probe's 3′ end and tolerated the mismatch at position M−18.

[0177] The probes used for HLA-B were: SB220 CCGCGCGCTCCAGCGTG SB246CCACTCCATGAGGTATTTCC SB229 CTCCAACTTGCGCTGGGA SB272 CGCCACGAGTCCGAGGAASB285 GTCGTAGGCGTCCTGGTC SB221 TACCAGCGCGCTCCAGCT SB197AGCAGGAGGGGCCGGAA SB127 CGTCGCAGCCATACATCCA SB187 GCGCCGTGGATAGAGCAASB188 GCCGCGAGTCCGAGGAC SB195 GACCGGAACACACAGATCTT

[0178] Experiments using these probes for typing HLA-B exon 2 wereperformed using reference cell lines. As with HLA-A, unambiguous results(not shown here) were obtained.

Example 10 CF Mutation Analysis—Probe and Array Design for ProbeElongation

[0179] This Example describes the design and application of a planararray of probes, displayed on color-encoded particles, these probesdesigned to display several—most frequently two selected basecompositions at or near their respective 3′ ends and designed to alignwith designated regions of interest within the CFTR target gene.

[0180] The CFTR gene sequence from Genebank (www.ncbi.nlm.nih.gov) wasused to design sixteen-mer probes for the multiplexed analysis of the 25CFTR mutations in the ACMG-CF mutation panel. Probe sequences weredesigned using PROBE 3.0 (http://www.genome.wi.mit.edu) and aligned withrespective exon sequences(http://searchlauncher.bcm.tmc.edu/seq-search/alignment.html).Oligonucleotides were designed to comprise 15 to 21 nucleotides, with a30-50% G+C rich.base composition and synthesized to contain a 5′ biotinTEG (Synthegen Tex.); to handle small deletions, the variable sequenceof the TEI region was placed at or within 3-5 positions of the probe's3′ terminus. Probe compositions are listed in the table below.

[0181] A combination of 17 either pure blue or blue-green stained beadswere used with CF mutation analysis. The 48 base long Human β-actin gene(Accession #X00351) was synthesized and used in each reaction as aninternal positive control. Sixteen base long complementary probes wereincluded on each array. The CFTR gene sequence from Genebank(www.ncbi.nlm.nih.gov) was used for probe design for analysis of 25 CFTRmutations in the ACMG-CF mutation panel. The probe sequences weredesigned by PROBE 3.0 (http://www.genome.wi.mit.edu). Each probesequence was aligned with respective exon sequences(http://searchlauncher.bcm.tmc.edu/seq-search/alignment.html).Oligonucleotides were synthesized with a 5′ biotin TEG (Synthegen Tex.)and coupled on the surface of beads in presence of 0.5 M NaCl. Beadswere immobilized on the surface of a chip by LEAPS.

[0182] Exon Mutations Sequence EXON MUTATIONS SEQUENCE  3 G85E CCC CTAAAT ATA AAA AGA TTC G85E-X CCC CTA AAT ATA AAA AGA TTT  4 1148 ATT CTCATC TCC ATT CCA A 1148-X ATT CTC ATC TCC ATT CCA G 621+1G>T TGT GTG CAAGGA AGT ATT AC 621+1G>T-X TGT GTG CAA GGA AGT ATT AA R117H TAG ATA AATCGC GAT AGA GC R117H-X TAG ATA AAT CGC GAT AGA GT  5 711+1G>T TAA ATCAAT AGG TAC ATA C TAA ATC AAT AGG TAC ATA A  7 R334W ATG GTG GTG AAT ATTTTC CG R334W-X ATG GTG GTG AAT ATT TTC CA R347P ATT GCC GAG TGA CCG CCATGC R347P-X ATT GCC GAG TGA CCG CCA TGG 1078delT CAC AGA TAA AAA CAC CACAAA 1078delT-X CAC AGA TAA AAA CAC CAC AA 1078delT-X-2 CAC AGA TAA AAACAC CAC A  9 A455E TCC AGT GGA TCC AGC AAC CG A455E-X TCC AGT GGA TCCAGC AAC CT 10 508 CAT AGG AAA CAC CAA AGA T 1507 CAT AGG AAA CAC CAA AF508 CAT AGG AAA CAC CAA T 11 1717-1G>A CTG CAA ACT TGG AGA TGT CC1717-1G>A CTG CAA ACT TGG AGA TGT CT 551D TTC TTG CTC GTT GAC 551D-X TTCTTG CTC GTT GAT R553 TAAAGAAATTCTTGCTCG R553X TAAAGAAATTCTTGCTCA R560ACCAATAATTAGTTATTCACC R560X ACCAATAATTAGTTATTCACG G542GTGTGATTCCACCTTCTC C G542X GTGTGATTCCACCTTCTC A INT-12 1898 AGG TAT TCAAAG AAC ATA C 1898-X AGG TAT TCA AAG AAC ATA T 2183deLA TGT CTG TTT AAAAGA TTG T 13 2183deLA-X TGT CTG TTT AAA AGA TTG C INT 14B 2789 CAA TAGGAC ATG GAA TAC 2789-X CAA TAG GAC ATG GAA TAC T INT16 3120 ACT TAT TTTTAC ATA C 3120-X ACT TAT TTT TAC ATA T 18 D1152 ACT TAC CAA GCT ATC CACATC D1152 ACT TAC CAA GCT ATC CAC ATG INT 19 3849+10kbC>T-WT1 CCT TTCAgg GTG TCT TAC TCG 3849+10kbC>T-M1 CCT TTC Agg GTG TCT TAC TCA 19 R1162AAT GAA CTT AAA GAC TCG R1162-X AAT GAA CTT AAA GAG TCA 3659delC-WT1 GTATGG TTT GGT TGA CTT GG 3659delCX-M1 GTA TGG TTT GGT TGA CTT  GTA3659delC-WT2 GTA TGG TTT GGT TGA CTT GGT A 3659delCX-M2 GTA TGG TTT GGTTGA CTT  GT A 20 W1282 ACT CCA AAG GCT TTC CTC W1282-X CT CCA AAG GCTTTC CTT 21 N1303K TGT TCA TAG GGA TCC AAG N1303K-X TGT TCA TAG GGA TCCAAG b β Actin AGG ACT CCA TGC CCA G

[0183] Probes were attached, in the presence of 0.5 M NaCl, todifferentially encoded beads, stained either pure blue or blue-greenBeads were immobilized on the surface of a chip using LEAPS. A synthetic48 base Human β-actin gene (Accession #X00351) was included in eachreaction as an internal positive control.

[0184] Array Design—In a preferred embodiment, the 25 CF mutations weredivided into four different groups so as to minimize sequence homologiesbetween members of each group. That is, mutations were sorted intoseparate groups so as to minimize overlap between probe sequences in anysuch group and thereby to minimize cross-hybridization under conditionsof multiplexed analysis. Each group, displayed on color-encoded beads,was assembled into a separate array. (Results for this 4-chip arraydesign are described in the following Example). Alternative robust arraydesigns also are disclosed herein.

Example 11 Multiplexed CF Mutation Analysis by Probe Elongation UsingREAD

[0185] Genomic DNA, extracted from several patients, was amplified withcorresponding probes in a multiplex PCR (mPCR) reaction using the methoddescribed in L. McCurdy, Thesis, Mount Sinai School of Medicine, 2000,which is incorporated by reference. This mPCR reaction uses chimericprimers tagged with a universal sequence at the 5′ end. Antisenseprimers were phosphorylated at the 5′ end (Synthegen, Tex.). Twentyeight amplification cycles were performed using a Perkin Elmer 9600thermal cycler, each cycle comprising a 10 second denaturation step at94° C. with a 48 second ramp, a 10 second annealing step at 60° C. witha 36 second ramp and a 40 second extension step at 72° C. with a 38second ramp, each reaction (50 μl) containing 500 ng genomic DNA, 1×PCRbuffer (10 mM Tris HCL, 50 mM KCL, 0.1% Triton X-100), 1.5 mM MgCl₂, 200μM each of PCR grade dNTPs and 5 units Taq DNA polymerase. Optimal probeconcentrations were determined for each probe pair. Followingamplification, products were purified to remove all reagents using acommercially available kit (Qiagen). DNA concentration was determined byspectrophotometric analysis.

[0186] PCR products were amplified with antisense 5′-phosphorylatedprimers. To produce single-stranded DNA templates, PCR reaction productswere incubated with 2.5 units of exonuclease in 1× buffer at 37° C. for20 min, followed by enzyme inactivation by heating to 75° C. for 10 min.Under these conditions, the enzyme digests one strand of duplex DNA fromthe 5′-phosphorylated end and releases 5′-phosphomononucleotides (J. W.Little, et al., 1967). Single-stranded targets also can be produced byother methods known in the art.

[0187] Single or pooled PCR products (20 ng each) were added to anannealing mixture containing 10 mM Tris-HCL (pH 7.4) 1 mM EDTA, 0.2 MNaCl, 0.1% Triton X-100. The annealing mixture was placed in contactwith the encoded array of bead-displayed CF probes (of Example 10) andincubated at 37-55° C. for 20 minutes. The extension mixture—containing3 U of Thermo Sequenase (Amersham Pharmacia Biotech N.J.), 1× enzymebuffer with either Fluorescein-labeled or TAMRA-labeled deoxynucleotide(dNTP) analogs (NEN Life Sciences) and 1 μmole of each type of unlabeleddNTP—was then added, and the elongation reaction was allowed to proceedfor 3 minutes at 60° C. The bead array was washed with deionized,sterilized water (dsH₂O) for 5-15 minutes. An image containing thefluorescence signal from each bead within the array was recorded using afluorescence microscope equipped with a CCD camera. Images were analyzedto determine the identity of each of the elongated probes. The resultsare shown in FIG. 15.

Example 12 Use of Covering Probes

[0188] Several SNPs have been identified within exon 10 of the CFTRgene. The polymorphisms in exon 10 are listed at the end of thisExample. The following nine SNPs have been identified in the sequence ofΔ508, the most common mutation in the CFTR gene (http://snp.cshl.org):

[0189] dbSNP213450 A/G

[0190] dbSNP180001 C/T

[0191] dbSNP1800093 G/T

[0192] 1648 A/G

[0193] dbSNP100092 C/G

[0194] dbSNP1801178 A/G

[0195] dbSNP1800094 A/G

[0196] dbSNP1800095 G/A

[0197] Probes are designed to accommodate all possible SNPs aresynthesized and coupled to color-encoded beads. The primers for targetamplification (described in Example 11) are also modified to take intoaccount all possible SNPs. The PCR-amplified target mediates theelongation of terminally matched probes. The information collected fromthe analysis is twofold: identification of mutations and SNPs.

[0198] Exon 10 Polymorphisms EXON 10 POLYMORPHISMS 1 cactgtagctgtactacctt ccatctcctc aacctattcc aactatctga atcatgtgcc 61 cttctctgtgaacctctatc ataatacttg tcacactgta ttgtaattgt ctcttttact 121 ttcccttgtatcttttgtgc atagcagagt acctgaaaca ggaagtattt taaatatttt 181 gaatcaaatgagttaataga atctttacaa ataagaatat acacttctgc ttaggatgat 241 aattggaggcaagtgaatcc tgagcgtgat ttgataatga cctaataatg atgggtttta 301 tttccagacttcaCttctaa tgAtgattat gggagaactg gagccttcag agggtaaaat 361 taagcacagtggaagaattt cattctgttc tcagttttcc tggattatgc ctggcaccat 421 taaagaaaatAtCAtctTtg gtgtttccta tgatgaatat agatacagaa gcgtcatcaa 481 agcatgccaactagaAgagG taagaaacta tgtgaaaact ttttgattat gcatatgaac 541 ccttcacactacccaaatta tatatttggc tccatattca atcggttagt ctacatatat 601 ttatgtttcctctatgggta agctactgtg aatggatcaa ttaataaaac acatgaccta 661 tgctttaagaagcttgcaaa cacatgaaat aaatgcaatt tattttttaa ataatgggtt 721 catttgatcacaataaatgc attttatgaa atggtgagaa ttttgttcac tcattagtga 781 gacaaacgtctcaatggtta tttatatggc atgcatatag tgatatgtgg t

Example 13 CF Mutation Analysis—On-Bead Probe Elongation with ModelSystem

[0199]FIG. 13 provides an overview of detection of CF gene mutationR117H. The target was amplified by PCR as described in Example 11. Two17-base probes variable at their 3′ ends were immobilized on color codedbeads. The target nucleic acid sequence was added along withTAMRA-labeled dCTP, unlabeled dNTPs and thermostable DNA polymerase.

[0200] Complementary 17-mer oligonucleotide probes variable at the 3′end were were synthesized by a commercial vendor (Synthegen Tex.) tocontain 5′ biotin attached by way of a 12-C spacer (Biotin-TEG) and werepurified by reverse phase HPLC. Probes were immobilized on color encodedbeads. Probes were attached to color-encoded beads. A synthetic 48-meroligonucleotide also was provided to contain either A,T,C or G at adesignated variable site, corresponding to a cystic fibrosis genemutation at exon 4 (R117H).

[0201] 1 μM of synthetic target was added to an annealing mixturecontaining 10 mM Tris-HCL (pH 7.4) 1 mM EDTA, 0.2 M NaCl, 0.1% TritonX-100. The annealing mixture was placed in contact with the encoded beadarray and incubated at 37° C. for 20 minutes. An elongation mixturecontaining 3 U of Thermo Sequenase (Amersham Pharmacia Biotech N.J.), 1×enzyme buffer with TAMRA-labeled deoxynucleotide (dNTP) analogs (NENLife Sciences) and 1 μM of each type of unlabeled dNTP was then added,and the elongation reaction was allowed to proceed for 3 minutes at 60°C. The bead array was then washed with dsH₂O for 5-15 minutes and animage containing the fluorescence signal from each bead within the arraywas recorded using a fluorescence microscope equipped with a CCD camera.Images were analyzed to determine the identity of each of the elongatedprobes. The signal was analyzed by capturing the image by a CCD cameraand comparing signal intensity between two probes that can be decoded bythe bead color. The wild-type type probe exactly matched the addedtarget and therefore yielded an elongation product, whereas noelongation was observed for the mutant probe. The results are shown inFIG. 16a.

Example 14 CF Mutation Analysis—PCR with Bead-Tagged Primers andIntegrated Detection

[0202] This example illustrates probe elongation on the surface of beadsin suspension, followed by assembly of and immobilization of beads onthe surface of a chip for image analysis. Oligonucleotides correspondingto CFTR gene mutation R117H were designed with variable 3′ ends (FIG.14) and were synthesized to contain a 5′ biotin-TEG with a 12 C spacer(Synthegen, Tex.). The probes were attached to blue stained beads asfollows: 2 μM of probe were added to a bead solution in 1×TE (100 mMTris-HCl, 10 mM EDTA), 500 mM NaCl and reacted for 45 min at roomtemperature. Beads were washed with 1×TE, 150 mM of NaCl for 3×, andsuspended in 50 μl of the same solution. One μl of each type of bead wasadded to PCR mix containing 1× buffer (100 mM Tris-HCl, pH. 9.0, 1.5 mMMgCl₂, 500 mM KCl), 40 μM Cy5-labeled dCTP (Amersham Pharmacia BiotechN.J.), and 80 μM of the other three types of dNTPs, and 3 U of Taq DNApolymerase (Amersham Pharmacia Biotech N.J.). Wild type complementarytarget (40 ng) was added to the PCR mix just before amplification.Eleven cycles of PCR amplification were performed in a Perkin Elmer 9600thermal cycler, each cycle consisting of denaturation for 30 s at 90°C., annealing for 30 s at 55° C., and elongation at 72° C. for 20 sAfter amplification, beads were washed four times by centrifugation in1×TE buffer and placed on the chip surface. Images were recorded as inprevious Examples and analyzed using the software described in WO01/98765. The results show specific amplification for beads coupled withthe wild-type probe, but no amplification for beads coupled with themutant probe. The results are shown in FIG. 16b.

[0203] This example demonstrates the integration of multiplexed PCRusing bead-tagged probes with subsequent assembly of beads on planarsurfaces for instant imaging analysis. In a preferred embodiment, amicrofluidically connected multicompartment device may be used fortemplate amplification as described here. For example, a plurality ofcompartments capable of permitting temperature cycling and housing, ineach compartment, one mPCR reaction producing a subset of all desiredamplicons may be used as follows: (1) perform PCR with different probepairs in each of four compartments, using encoded bead-tagged primers asdescribed in this Example; (2) following completion of all PCRreactions, pool the amplicon-displaying beads; (3) assemble randomarray; and (4) record image and analyze the data. Array assembly may beaccomplished by one of several methods of the prior art including LEAPS.

Example 15 CF Mutation Analysis—One Step Annealing and Elongation inTemperature-Controlled Reactor

[0204] Genomic DNA, extracted from several patients, was amplified withcorresponding primers in a multiplexed PCR (mPCR) reaction, as describedin Example 11. Following amplification, products were purified to removeall reagents using a commercially available kit (Qiagen). DNAconcentration was determined by spectrophotometric analysis. Single orpooled PCR products (20 ng each) were added to an annealing mixturecontaining 10 mM Tris-HCL (pH 7.4) 1 mM EDTA, 0.2 M NaCl, 0.1% TritonX-100. The annealing mixture was mixed with elongation mixturecontaining 3 U of Thermo Sequenase (Amersham Pharmacia Biotech, N.J.),1× enzyme buffer with either fluorescein-labeled or TAMRA-labeleddeoxynucleotide (dNTP) analogs (NEN Life Sciences) and 1-10 μmole ofeach type of unlabeled dNTP and placed in contact with an array ofoligonucleotide probes displayed on a color-encoded array.Oligonucleotides were designed and synthesized as in previous Examples.The annealing and elongation reactions were allowed to proceed in atemperature controlled cycler. The temperature steps were as follows:three minutes each at 65° C., 60° C., 55° C., 50° C. and 45° C., with aramp between temperatures seconds. The bead array was then washed withdsH₂O for 5 to 15 min. and an image containing the fluorescence signalfrom each bead within the array was recorded using a fluorescencemicroscope equipped with a CCD camera. Images were analyzed to determinethe identity of each of the elongated probes. Typical results are shownin FIG. 17.

Example 16 Pooling of Covering Probes

[0205] To analyze designated polymorphisms, 20-mer oligonucleotideelongation probes of 30-50% G+C base composition were designed tocontain a variable site (G/T) at the 3′ end, to be aligned with thedesignated polymorphic site. Two non-designated polymorphic sites wereanticipated at position 10 (C/A) and at 15 (T/G). A summary of thedesign follows:

[0206] Wild-type probe sequence:

[0207] Oligo 1: “G” at position 20, “C” at 10, and “T” at 15.

[0208] Oligo 2: “G” at position 20, “C” at 10, and “G” at 15.

[0209] Oligo 3: “G” at position 20, “A” at 10, and “T” at 15.

[0210] Oligo 4: “G” at position 20, “A” at 10, and “G” at 15.

[0211] Mutant Probe Sequence:

[0212] Oligo 1: “T” at position 20, “C” at 10, and “T” at 15.

[0213] Oligo 2: “T” at position 20, “C” at 10, and “G” at 15.

[0214] Oligo 3: “T” at position 20, “A” at 10, and “T” at 15.

[0215] Oligo 4: “T” at position 20, “A” at 10, and “G” at 15.

[0216] All of the probes were pooled and attached to a single type ofcolor-coded bead using protocols of previous Examples. Whensingle-stranded target is added to these beads displaying pooled probes,one of the probes will yield elongation product as long as it isperfectly aligned with the designated polymorphism.

Example 17 Designated Polymorphisms in Heterozygous and HomozygousConfigurations

[0217] To distinguish between heterozygous and homozygousconfigurations, the design of the previous Example is augmented tocontain a second set of probes to permit the identification of the C/Adesignated polymorphism aligned with the probes' 3′ ends, and to permitcalling of heterozygous versus homozygous mutations.

[0218] As in the previous example, two non-designated polymorphic sitesare anticipated at positions 10 (C/A) and 15 (T/G). A summary of thedesign follows:

[0219] Set #1:

[0220] Oligo 1: “C” at position 20, “C” at 10, and “T” at 15.

[0221] Oligo 2: “C” at position 20, “C” at 10, and “G” at 15.

[0222] Oligo 3: “C” at position 20, “A” at 10, and “T” at 15.

[0223] Oligo 4: “C” at position 20, “A” at 10, and “G” at 15.

[0224] Set #2:

[0225] Oligo 5: “A” at position 20, “C” at 10, and “T” at 15.

[0226] Oligo 6: “A” at position 20, “C” at 10, and “G” at 15.

[0227] Oligo 7: “A” at position 20, “A” at 10, and “T” at 15.

[0228] Oligo 8: “A” at position 20, “A” at 10, and “G” at 15.

[0229] Oligonucleotides from set #1 are pooled and attached to a singletype of color (e.g. green) coded bead using protocols of previousExamples. Oligonucleotides from set # 2 were pooled and attached to ascond type of color (e.g. orange) coded bead using protocols of previousExamples. Beads were pooled and immobilized on the surface of chip asdescribed earlier. Next, target was introduced, and on-chip reactionsperformed as described in previous Examples. If probes on green beadsonly are elongated, the individual has a normal (or wild-type) allele.If probes on orange beads only are elongated, the individual ishomozygous for the mutation. I If probes on green as well as origanbeads are elongated, the individual is heterozygous for that allele.This design is useful for the identification of known and unknownmutations.

Example 18 Confirmatory Sequencing (“Resequencing”)

[0230] The design of the present invention can be used for re-sequencingof a specific area. This test can be used when on-chip probe elongationreaction requires confirmation, as in the case of reflex tests for1506V, 1507V, F508C and 7T in the CF mutation panel. The sequence inquestion, here 20 bases to 30 bases in length, is sequenced on-chip bymultiplexed interrogation of all variable sites. This is accomplished bydesigning specific probes for ambiguous locations, and by probe-poolingas described in Examples 16 and 17.

Example 19 Elongation with One Labeled dNTP and Three Unlabeled dNTPs

[0231] By way of incorporating at least one labeled dNTP, all elongationproducts are detected in real-time and identified by their associationwith coded solid phase carriers. Using assay conditions described inconnection with Examples 6 and 7, tetramethylrhodamine-6-dCTP andunlabeled dATP, dTTP and dGTP were provided in an elongation reaction toproduce a fluorescently labeled elongation product as illustrated FIG.18. Other dye labeling of dNTPs (as in BODIPY-labeled dUTP andCy5-labeled dUTP) may be used. Similarly, any other labeled dNTP can beused. The length of the elongation product depends on the amount oflabeled dNTP tolerated by the DNA polymerase. Available enzymesgenerally exhibit a higher tolerance for strand-modifying moieties suchas biotin and digoxigenin which may then be reacted in a second stepwith labeled avidins or antibodies to accomplish indirect labeling ofelongation procucts. When using these small molecules, elongationproducts measuring several hundred bases in length are produced.

Example 20 Extension with One Labeled ddNTP, Three Unlabeled dNTPs

[0232] TAMRA-labeled ddCTP may be incorporated to terminate theextension reaction, as illustrated in FIG. 19. On-chip reactions usingTAMRA-labeled ddCTP were performed as described in Examples 6 and 7. Ina reaction mixture containing TAMRA-ddCTP and unlabeled dTTP, dATP anddGTP, following annealing of the target to the matching probe, theextension reaction terminates when it completes the incorporation of thefirst ddCTP. This may occur with the very first base incorporated,producing a single base extension product, or it may occur after anumber of unlabeled dNTPs have been incorporated.

Example 21 Elongation with Four Unlabeled dNTPs, Detection byHybridization of Labeled Probe

[0233] Probes are elongated using a full set of four types of unlabeleddNTPs, producing, under these “native” conditions for the polymerase,elongation products measuring several hundred bases in length, limitedonly by the length of the annealed template and on-chip reactionconditions. The elongation product is detected, following denaturationat high temperature, in a second step by hybridization with a labeledoligonucleotide probe whose sequence is designed to be complementary toa portion of the elongation product This process is illustrated in FIG.20.

Example 22 Elongation with Four Unlabeled dNTPs, Detection via LabeledTemplate

[0234] As with standard protocols in routine use in multiplexedhybridization assays, the DNA target to be analyzed can itself belabeled in the course of PCR by incorporation of labeled probes. Underconditions such as those described in Examples 6 and 7, a labeled targetis annealed to probes. Matching probes are elongated using unlabeleddNTPs. Following completion of the elongation reaction, detection isperformed by setting the temperature (T_(det)) to a value above themelting temperature (T_(non-match)) of the complex formed by target andnon-matched probe, but below the melting temperature (T_(match)) of thecomplex formed by target and matched, and hence elongated, probe. Thelatter complex, displaying a long stretch of duplex region,will besignificantly more stable than the former so that(T_(non-match))<T<(T_(match)). Typical values for T are in the range of70° C. to 80° C. Under these conditions, only the complex formed bytarget and elongated probe will stable, while the complex formed bytarget and non-matching probe, and hence the fluorescence signal fromthe corresponding solid phase carrier, will be lost. That is, incontrast to other designs, it is the decrease of signal intensityassociated with the non-matching probe which is detected, rather thanthe increase in intensity associated the matching probe. FIG. 21illustrates the design which eliminates the need for labeled dNTPs orddNTPs. This is useful in the preferred embodiments of this invention,where labeled dNTPs or ddNTPs can absorb non-specifically to encodedparticles, thereby increasing the background of the signal anddecreasing the discriminatory power of the assays. In addition, by usinga labeled target, this protocol is directly compatible with methods ofpolymorphism analysis by hybridization of sequence-specificoligonucleotides.

Example 23

[0235] Real-Time On-chip Signal Amplification

[0236] A standard temperature control apparatus used with a planargeometry such as that illustrated in FIG. 22 permits the application ofprogrammed temperature profiles to a multiplexed extension of SSPs.Under conditions of Examples 6 and 7, a given template mediates theelongation of one probe in each of multiple repeated“denature-anneal-extend” cycles. In the first cycle, a target moleculebinds to a probe and the probe is elongated or extended. In the nextcycle, the target molecule disassociates from the first probe in the“denature” phase (at a typical temperature of 95° C.), then anneals withanother probe molecule in the “anneal” phase (at a typical temperatureof 55° C.) and mediates the extension of the probe in the “extend” phase(at a typical temperature of 72° C.). In N cycles, each templatemediates the extension of N probes, a protocol corresponding to linearamplification (FIG. 30). In a preferred embodiment of this invention, inwhich planar arrays of encoded beads are used to display probes in amultiplexed extension reaction, a series of temperature cycles isapplied to the reaction mixture contained between two planar, parallelsubstrates. One substrate permits direct optical access and directimaging of an entire array of encoded beads. The preferred embodimentprovides for real-time amplification by permitting images of the entirebead array to be recorded instantly at the completion of each cycle.

[0237] Genomic, mitochondrial or other enriched DNA can be used fordirect detection using on-chip linear amplification without sequencespecific amplification. This is possible when an amount of DNAsufficient for detection is provided in the sample. In the bead arrayformat, if 10⁴ fluorophores are required for detection of signal fromeach bead, 30 cycles of linear amplification will reduce the requisitenumber to ˜300. Assuming the use of 100 beads of the requisite typewithin the array, the requisite total number of fluorophores would be˜10⁵, a number typically available in clinical samples. For example,typical PCR reactions for clinical molecular typing of HLA are performedwith 0.1 to 1 μg of genomic DNA. One μg of human genomic DNA correspondsto approximately 10⁻¹⁸ moles, thus, 6×10⁵ copies of the gene of interestThis small amount of sample required by the miniaturized bead arrayplatform and on-chip amplification makes the direct use of pre-PCRsamples possible. This not only simplifies sample preparation but, moreimportantly, eliminates the complexity of multiplexed PCR, frequently arate limiting step in the development of multiplexed genetic analysis.

Example 24 Construction of a Probe Library for Designated and UnselectedPolymorphisms for CF Mutation Analysis

[0238] To increase the specificity of elongation probes and avoid falsepositives, elongation probes were designed to accommodate all knownpolymorphisms present in a target sequence. In addition, PCR primerswere designed taking into consideration designated and non-designatedpolymorphisms.

[0239] The G/C mutation at position 1172. of R347P on Exon 7 within theCFTR gene, one of 25 mutations within the standard population carrierscreening panel for cystic fibrosis, was selected as a designatedpolymorphism. There are 3 CF mutations within Exon 7 included in themutation panel for general population carrier screening(http://www.faseb.org/genetics/acmg). A polymorphism G/T/A at the samesite has been reported (http://www.genet.sickkids.on.ca/cftr), and inaddition, non-designated polymorphisms have been reported at positions1175, 1178, 1186, 1187 and 1189. All of these polymorphisms caninterfere with desired probe elongation.

[0240] The construction of a set of degenerate probes for eMAP isillustrated below for R347P (indicated by the bold-faced G) which issurrounded by numerous non-designated polymorphisms, indicated bycapital letters: 5′                       3′ Normal Target Sequence forElongation: Gca Tgg Cgg tca ctC GgC a Degenerate Elongation Probe Set:Ngt Ycc Ycc agt gaY RcY t 3′                       5′+TZ,1 41

[0241] where N=a, c, g or t; R (puRines)=a or g and Y (pYrimidines)=c ort, implying a degeneracy of 128 for the set.

[0242] Primer Pooling for Mutation Analysis—The principal objective inthe construction of a degenerate set is to provide at least one probesequence to match the target sequence sufficiently closely to ensureprobe annealing and elongation. While this is always attainable inprinciple by providing the entire set of possible probe sequencesassociated with the designated polymorphism, as in the preferred mode ofconstructing covering sets, the degree of degeneracy of that set, 128 inthe example, would lead to a corresponding reduction in assay signalintensity by two orders of magnitude if all probes were to be placedonto a single bead type for complete probe pooling. Splitting poolswould improve the situation by distributing the probe set over multiplebead types, but only at the expense of increasing array complexity.

[0243] First, the probe pool was split into a minimum of two or morepools, each pool providing the complementary composition, at probeposition M (i.e., the probe's 3′ terminus), for each of the possiblecompositions of the designated polymorphic site. In the example, foursuch pools are required for a positive identification of the designatedtarget composition. Next, non-designated polymorphic sites were examinedsuccessively in the order of distance from the designated site. Amongthese, positions within the TEI region are of special importance toensure elongation. That is, each pool is constructed to contain allpossible probe compositions for those non-designated sites that fallwithin the TEI region. Finally, as with the construction of degenerateprobes for cloning and sequencing of variable genes, the degeneracy ofthe set is minimized by placing neutral bases such as inosine into thoseprobe positions which are located outside the TEI region provided theseare known never to be juxtaposed to G in the target. In the example,non-designated polymorphisms in probe positions M−16 and M−18 qualify.That is, the minimal degeneracy of each of the four pools would increaseto four, producing a corresponding reduction in signal intensity. As anempirical guideline, signal reduction preferably will be limited to afactor of eight.

[0244] In total, four pools, each uniquely assigned to one bead type andcontaining eight degenerate probe sequences, will cover the targetsequence. These sequences are analogous to those shown below for poolsvariable at M:

[0245] Probe pool for CF mutation R347P Probe pool for CF mutation R347PR347P Cgt Acc Gcc agt gaG GgC 3′                       5′ POOL 1 Cgt AccGcc agt gaG IgI Cgt Acc Gcc agt gaC IgI Cgt Acc Ccc agt gaG IgI Cgt AccCcc agt gaC IgI Cgt Tcc Gcc agt gaG IgI Cgt Tcc Gcc agt gaC IgI Cgt TccCcc agt gaG IgI Cgt Tcc Ccc agt gaC IgI POOL 2 Ggt Acc Gcc agt gaG IgIGgt Acc Gcc agt gaC IgI Ggt Acc Ccc agt gaG IgI Ggt Acc Ccc agt gaC IgIGgt Tcc Gcc agt gaG IgI Ggt Tcc Gcc agt gaG IgI Ggt Tcc Ccc agt gaG IgIGgt Tcc Ccc agt gaC IgI POOL 3 Agt Acc Gcc agt gaG IgI Agt Acc Gcc agtgaC IgI Agt Acc Ccc agt gaG IgI Agt Acc Ccc agt gaC IgI Agt Tcc Gcc agtgaG IgI Agt Tcc Gcc agt gaC IgI Agt Tcc Ccc agt gaG IgI Agt Tcc Ccc agtgaC IgI POOL 4 Tgt Acc Gcc agt gaG IgI Tgt Acc Gcc agt gaC IgI Tgt AccCcc agt gaG IgI Tgt Acc Ccc agt gaC IgI Tgt Tcc Gcc agt gaG IgI Tgt TccGcc agt gaC IgI Tgt Tcc Ccc agt gaG IgI Tgt Tcc Ccc agt gaC IgI

[0246] In general, the type of non-designated polymorphisms on theantisense strand may differ from that on the sense strand, and it maythen be advantageous to construct degenerate probe sets for theantisense strand. As with the construction of degenerate elongationprobes, degenerate hybridization probe sets may be constructed byanalogous rules to minimize the degeneracy.

Example 25 “Single Tube” CF Mutation Analysis by eMAP

[0247] This example is concerned with methods and compositions forperforming an eMAP assay, wherein the annealing and elongation stepsoccur in the reactor. This embodiment is useful because it obviates theneed for sample transfer between reactors as well as purification orextraction procedures, thus simplifying the assay and reducing thepossibility of error. A non-limiting exemplary protocol follows.

[0248] Genomic DNA extracted from several patients was amplified withcorresponding primers in a multiplex PCR (mPCR) reaction. The PCRconditions and reagent compositions were as follows.

[0249] PRIMER DESIGN: Sense primers were synthesized without anymodification and antisense primers with “Phosphate” at the 5′ end.Multiplex PCR was performed in two groups. Group one amplificationincludes exon 5, 7, 9, 12, 13, 14B, 16, 18 and 19. Amplifications forgroup 2 includes primers for exon 3, 4, 10, 11, 20, 21 and intron 19.The 5′ phosphate group modification on exon 5, 7, and 11 was included onforward primer to use antisense target for probe elongation. While sensetarget was used for all other amplicons by placing phosphate group onreverse primer.

[0250] PCR Master Mix Composition For 10 ul reaction/sample: ComponentsVolume (μl) 10X PCR buffer 1.0 25 mM MgCl₂ 0.7 dNTPs (2.5 mM) 2.0 Primermix (Multiplex 10x) 1.5 Taq DNA polymerase 0.3 ddH2O 1.5 DNA 3.0 Total10 PCR Cycling 94° C. 5 min, 94° C. 10 sec., 60° C. 10 sec., 72° C. 40sec 72° C. 5 min., Number of cycles: 28-35

[0251] The reaction volume can be adjusted according to experimentalneed. Amplifications are performed using a Perkin Elmer 9600 thermalcycler. Optimal primer concentrations were determined for each primerpair. Following amplifications, 5 ul of the product was removed for gelelectrophoresis. Single stranded DNA targets were generated as follows:Two microliters of exonuclease was added to 5 μl of PCR product,incubated at 37° C. for 15 minutes and enzyme was denatured at 80° C.for 15 minutes. After denaturation, 1 μl of 10× exonuclease buffer wasadded with 1 μl of λ exonuclease (5 U/μl) and incubated at 37° C. for 20minutes and the reaction was stopped by heating at 75° C. for 10minutes.

[0252] On Chip Elongation

[0253] Wild type and mutant probes for 26 CF mutations were coupled onthe bead surface and assembled on the chip array. The probes were alsodivided into two groups. A third group was assembled for reflex testincluding 5T/7T/9T polymorphisms. Elongation Group 1, total 31 groups onthe chip surface. Bead cluster # Mutation  1 G85E-WT  2 G85E-M  3 621 +1G > T-WT  4 621 + 1G > T-M  5 R117H-WT  6 R117H-M  7 β Actin  81148T-WT  9 1148T-M 10 508-WT 11 F508 12 I507 13 G542X-WT 14 G542X-M 15G551D-WT 16 G551D-M 17 R553X-WT 18 R553X-M 19 BIOTIN 20 1717-1G > A-WT21 1717-1G > A-M 22 R560T-WT 23 R560T-M 24 3849 + 10kbT-WT 25 3849 +10kbT-M 26 W1282X-WT 27 W1282X-M 28 N1303K-WT 29 N1303K-M 30 OLIGO-CCluster # Mutation Elongation Group 2, total 28 groups on the chipsurface.  1 711 + IG > T-WT  2 711 + 1G > T-M  3 R334W-WT  4 R334W-M  51078delT-WT  6 1078delT-M  7 β Actin  8 R347P-WT  9 R347P-M 10 A455E-WT11 A455E-M 12 1898 + 1G > A-WT 13 1898 + 1G > A-WT 14 2184delA-WT 152184delA-M 16 2789 + 5G-WT 17 2789 + 5G-M 18 BIOTIN 19 3120 + 1G > A-WT20 3120 + 1G > A-WT 21 R1162X-WT 22 R1162X-M 23 3659delC-WT 243659delC-M 25 D1152-WT 26 D1152-M 27 OLIGO-C mPCR group 2: ElongationGroup 3, total 6 groups  1 β Actin  1 Oligo C  2 5T  3 7T  4 9T  5Biotin

[0254] Elongation reaction buffer has been optimized for use in uniplexand/or multiplex target elongation assays and composed of, Tris-HCL (pH8.5) 1.2 mM, EDTA 1 uM, DTT 10 μM, KCl 1 μM, MgCl₂ 13μM,_(—)2-Mercaptoethanol 10 μM, Glycerol 0.5%, Tween-20 0.05%, andNonidet 0.05%. Ten microliters of elongation reaction mixture was addedon each chip containing 1× Reaction buffer 0.1 μM of Labeled dNTP, 1.0μM of dNTPs mix, 3 U of DNA polymerase and 5 μl (˜5 ng) of target DNA(patient sample). The reaction mix was added on the chip surface andincubated at 53° C. for 15 min and then at 60° C. for 3 min. The chipwas washed with wash buffer containing 0.01% SDS, covered with a cleancover slip and analyzed using a Bioarray Solutions imaging system.Images are analyzed to determine the identity of each of the elongatedprobes.

Example 26 CF Mutation Analysis—Single Tube Single Chip-One StepElongation

[0255] Probes for 26 CF mutations and controls were coupled on thesurface of 51 types of beads. Probe coupled beads were assembled on thesurface of a single chip. Genomic DNA was extracted from severalpatients and was amplified with corresponding primers in a multiplexedPCR (mPCR) reaction, as described in the previous example. Followingamplification, single stranded DNA products were produced using λexonuclease. Single or pooled PCR products (˜5 ng) were added to areaction mixture containing reaction buffer, deoxynucleotide (dNTP)analogs (NEN Life Sciences), each type of unlabeled dNTP, and DNApolymerase (Amersham Pharmacia Biotech, N.J.). The annealing/elongationreaction was allowed to proceed in a temperature controlled cycler. Thetemperature steps were as follows: 20 minutes at 53° C., and 3 minutesat 60° C. The bead array was then washed with dsH₂O containing 0.01% SDSfor 5 to 15 minutes. An image containing the fluorescent signal formeach bead within the array was recorded using a fluorescence microscopeand a CCD camera. Images were analyzed to determine the identity of eachof the elongated probes.

[0256] The composition of bead chip containing 26 CF mutations isprovided below. Elongation Group 4, total 51 groups Cluster # Mutation 1β Actin 2 G85E-WT 3 G85E-M 4 621 + 1G > T-WT 5 621 + 1G > T-M 6 R117H-WT7 R117H-M 8 1148T-WT 9 1148T-M 10 711 + 1G > T-WT 11 711 + 1G > T-M 12A455E-WT 13 A455E-M 14 508-WT 15 F508 16 I507 17 R533-WT 18 R533-M 19G542-WT 20 G542-M 21 G551D-WT 22 G551D-M 23 R560-WT 24 R560-M 25 1898 +1G-WT 26 1898 + 1G-M 27 2184de1A-WT 28 2184de1A-M 29 2789 + 5G > A-WT 302789 + 5G > A-M 31 3120 + 1G-WT 32 3120 + 1G-WT 33 D1152-WT 34 D1152-M35 R1162-WT 36 R1162-M 37 OLIGO-C 38 W1282X-WT 39 W1282-M 40 N1303K-WT41 N1303-M 42 R334-WT 43 R334-M 44 1078delT-WT 45 1078delT-M 463849-10kb-WT 47 3849-10kb-M 49 1717-1G > A-WT 50 1717-1G > A-WT 51Biotin

Example 27 Identification of Three or More Base Deletions and/orInsertions by eMAP

[0257] Elongation was used to analyze mutations with more than 3 basedeletions or insertions. Probes were designed by placing mutant bases3-5 base before 3′ end. The wild type probes were designed to eitherinclude or exclude mutant bases (terminating before mutations). Thefollowing is an example of mutations caused by a deletion of ATCTCand/or insertion of AGGTA. The probe designs are as follows:

[0258] 1. WT1— - - - ATCTCgca

[0259] 2. WT2— - - -

[0260] 3. M1— - - - gca (deletion only)

[0261] 4. M2— - - - AGGTAgca (deletion and insertion)

[0262] Wild type probes were either coupled on the surface ofdifferentially encoded beads or pooled as described in this invention.Probes for mutation 1 (M1: deletion) and 2 (M2: insertion) were coupledon different beads. Both wild type probes provide similar information,while the mutant probes can show the type of mutation identified in aspecific sample.

Example 28 Hairpin Probes

[0263] In certain embodiments of this invention, bead-displayed primingprobes form hairpin structures. A hairpin structure may include asequence fragment at the 5′ end that is complementary to the TEI regionand the DA sequence, as shown in FIG. 23. During a competitivehybridization reaction, the hairpin structure opens whenever the DAregion preferentially hybridizes with the target sequence. Under thiscondition, the TEI region will align with the designated polymorphicsite and the elongation reaction will occur. The competitive nature ofthe reaction can be used to control tolerance level of probes.

Example 29 Analysis of Cystic Fibrosis and Ashkenazi Jewish DiseaseMutations by Multiplexed Elongation of Allele Specific OligonucleotidesDisplayed on Custom Bead Arrays

[0264] A novel assay for the high throughput multiplexed analysis ofmutations has been evaluated for ACMG+ panel of Cystic Fibrosismutations. In addition, an Ashkenazi Jewish disease panel also -has beendeveloped to detect common mutations known to cause Tay-Sachs, Canavan,Gaucher, Niemann-Pick, Bloom Syndrome, Fancomi Anemia, FamilialDysautonomia, and mucolipodosis IV.

[0265] In elongated-mediated multiplexed analysis of polymorphisms(eMAP), allele specific oligonucleotides (ASO) containing variable 3′terminal sequences are attached to color-encoded beads which are in turnarrayed on silicon chips. Elongation products for normal and mutantsequences are simultaneously detected by instant imaging of fluorescencesignals from the entire array.

[0266] In this example, several hundred clinical patient samples wereused to evaluate ACMG CF bead chips. As shown in FIG. 24, the assaycorrectly scored all of the mutations identified by standard DNAanalysis.

[0267] In summary, a multiplexed elongation assay comprising customizedbeads was used to study mutations corresponding to ACMG+ and Ashkenazidisease panels. The customized beads can be used for DNA and proteinanalysis. The use of these customized beads are advantageous for severalreasons including (1) instant imaging—the turnaround time for the assayis within two hours (2) automated image acquisition and analysis (3)miniaturization, which means low reagent consumption, and (4) thebeadchips are synthesized using wafer technology, so that millions ofchips can be mass-produced, if desired.

We claim
 1. A method of concurrent determination of nucleotide composition at designated polymorphic sites located within one or more target nucleotide sequences, said method comprising the following steps: (a) providing one or more sets of probes, each probe capable of annealing to a subsequence of said one or more target nucleotide sequences located within a range of proximity to a designated polymorphic site; (b) contacting the set of probes with said one or more target nucleotide sequences so as to permit formation of hybridization complexes by placing an interrogation site within a probe sequence in direct alignment with the designated polymorphic site; (c) for each hybridization complex, determining the presence of a match or a mismatch between the interrogation site and a designated polymorphic site; and (d) determining the composition of the designated polymorphic site.
 2. The method of claim 1 wherein said one or more target nucleotide sequences are produced in a multiplex PCR reaction using one or more primer sets.
 3. The method of claim 2 wherein said primers sets are degenerate primer sets.
 4. The method of claim 1 wherein said targets are fragments of genomic DNA.
 5. The method of claim 1 wherein said targets are fragments of cDNA.
 6. The method of claim 1 wherein one or more sets of probes are spatially encoded on a substrate.
 7. The method of claim 1 wherein one or more sets of probes are immobilized on encoded microparticles.
 8. The method of claim 7 wherein the encoded microparticles are assembled into a random encoded array.
 9. The method of claim 1 wherein each probe contains a terminal elongation initiation region capable of initiating an elongation or extension reaction.
 10. The method of claim 9 wherein the reaction is catalyzed by a polymerase lacking 3′→5′ exonuclease activity.
 11. The method of claim 1 wherein step (c) comprises adding one or more deoxynucleotide triphosphates.
 12. The method of claim 11 further comprising using a polymerase capable of extending or elongating probes.
 13. The method of claim 12 wherein the polymerase lacks 3′→5′ exonuclease activity.
 14. The method of claim 11 wherein at least one of the deoxy nucleotide triphosphates is labeled so as to generate an optically detectable signature associated with the elongation product.
 15. The method of claim 1 wherein an optical label is attached to one or more probes by annealing to the probes a fluorescently labeled target to form a fluorescent hybridization complex.
 16. The method of claim 15 further comprising using a polymerase capable of extending or elongating probes displaying a match by addition of one or more deoxynucleotide triphosphates to form an elongated hybridization complex.
 17. The method of claim 16 further comprising identifying elongation products by detecting the stability of optical signatures under conditions in which temperature is set to a value above the melting temperature of any hybridization complex formed by target and non-matched probe but below the melting temperature of any extended hybridization complex formed by target and elongated probe.
 18. The method of claim 15 wherein one or more probes from the set of probes are immobilized on encoded microparticles and a change in optical signature is detected.
 19. The method of claim 15 wherein one or more probes from the set of probes are immobilized on encoded microparticles which are arranged in random encoded arrays.
 20. The method of claim 19 wherein the arrays are arranged in a spatially encoded manner.
 21. The method of claim 15 wherein the change in optical signature is detected and particle identity is determined.
 22. A method of sequence-specific amplification of assay signals produced in the analysis of a nucleic acid sequence of interest in a biological sample, comprising the following steps: (a) providing a set of immobilized probes capable of forming a hybridization complex with the sequence of interest; (b) contacting said set of immobilized probes with said biological sample containing said sequence of interest under conditions which permit the sequence of interest to anneal to at least one of the immobilized probes to form a hybridization complex; (c) contacting said hybridization complex with a polymerase to allow elongation or extension of the probes contained within said hybridization complex; (d) converting elongation or extension of the probes into an optical signal; and (e) recording said optical signal from the set of immobilized probes in real time.
 23. The method of claim 22 further comprising performing one or more cycles, each cycle comprising “annealing-extending/elongating-detecting-denaturing” steps, wherein each cycle results in the increase of the number of extended or elongated probes in arithmetic progression.
 24. The method of claim 23 comprising the steps of: (a) setting a first temperature favoring the formation of a hybridization complex; (b) setting a second temperature favorable to polymerase-catalyzed extension; (c) converting extension or elongation into optical signal; (d) recording/imaging optical signals/signatures from all immobilized probes; and (e) setting a third temperature so as to ensure denaturation of all hybridization complexes.
 25. A method of forming a covering probe set for the concurrent interrogation of a designated polymorphic site located in one or more target nucleic acid sequences comprising the steps of: (a) determining the sequence of an elongation probe capable of alignment of the interrogation site of the probe with a designated polymorphic site; (b) further determining a complete set of degenerate probes to accommodate all non-designated as well as non-selected designated polymorphic sites while maintaining alignment of the interrogation site of the probe with the designated polymorphic site; and (c) reducing the degree of degeneracy by removing all tolerated polymorphisms.
 26. The method of claim 25 wherein the covering set contains at least two probes with different interrogation site composition per designated site.
 27. The method of claim 25 wherein the reduction of complexity in step (c) is accomplished by probe pooling.
 28. A method of identifying polymorphisms at one or more designated sites on one or more target nucleotides, the method comprising (a) providing one or more probes capable of interrogating said designated sites; (b) forming an elongation product by elongating one or more probes designed to interrogate a designated site; and (c) determining the compositions at said two or more sites.
 29. The method of claim 28 further comprising forming a hybridization complex by annealing to the elongation product a second probe designed to interrogate a second designated site.
 30. A method for identifying polymorphisms at one or more designated sites within a target polynucleotide sequence, the method comprising (a) providing one or more probes capable of interrogating said designated sites; (b) assigning a value to each such designated site while accommodating non-designated polymorphic sites located within a range of proximity to each such polymorphism.
 31. The method of claim 30 wherein the homology between the probes and the target sequence is analyzed by multiplexing.
 32. A method for determining polymorphism at one or more designated sites of a target nucleotide sequence, the method comprising the steps of providing one or more pairs of probes capable of detecting deletions wherein the deletions are placed either at the 3′ terminus of the probe or within 3-5 bases of the 3′ terminus.
 33. A method of identifying polymorphisms at two or more designated sites of a target nucleotide sequence, the method comprising (a) selecting a multiplicity of designated polymorphic sites to permit allele assignment; (b) providing two or more probes capable of concurrent interrogation of the multiplicity of designated sites; (c) assigning a value to each such designated site; and (d) combining said values to determine the identity of an allele or group of alleles while accommodating non-designated sites near said designated polymorphisms.
 34. A method for determining a polymorphism at one or more designated sites in a target polynucleotide sequence, the method comprising providing a probe set for such designated sites and grouping said probe set in different probe subsets according to the terminal elongation initiation of each probe.
 35. The method of claim 34 further comprising the step of multiplexing said probe set, measuring each probe in the probe set without interference from the other probes in the probe set and changing the allele matching pattern of a target polynucleotide sequence to include alleles that are tolerated by a probe set.
 36. The method of claim 35 wherein the step of changing the allele matching pattern of a target polynucleotide sequence comprises pooling one or more probe sets to include matched alleles.
 37. The method of claim 36 wherein the step of changing the allele matching pattern of a target polynucleotide sequence comprises the step of comparing the signal intensities produced by the probe set.
 38. The method of claim 37 further comprising the step of separating the terminal elongation initiation region and duplex anchoring region on the probe set.
 39. A method for the concurrent interrogation of a multiplicity of polymorphic sites comprising the step of conducting a multiplexed elongation assay by applying one or more temperature cycles to achieve linear amplification of such target.
 40. A method for the concurrent interrogation of a multiplicity of polymorphic sites comprising the step of conducting a multiplexed elongation assay by applying a combination of annealing and elongation steps under temperature-controlled conditions.
 41. A method of concurrent interrogation of nucleotide composition at S polymorphic sites, P sub S:={c sub P (s); 1<=s<=S} located within one or more contiguous target sequences, said method assigning to each c sub P one of a limited set of possible values by performing the following steps: (a) providing a set of designated immobilized oligonucleotide probes, also known as elongation probes, each probe capable of annealing in a preferred alignment to a subsequence of the target located proximal to a designated polymorphic site, the preferred alignment placing an interrogation site within the probe sequence in direct juxtaposition to the designated polymorphic site, the probes further containing a terminal elongation initiation (TEI) region capable of initiating an elongation or extension reaction; (b) permitting the one or more target sequences to anneal to the set of immobilized oligonucleotide probes so as form probe-target hyrbdization complexes; and (c) for each probe-target hybridization complex, calling a match or a mismatch in composition between interrogation site and corresponding designated polymorphic site.
 42. The method of claim 41, wherein probes are immobilized in a spatially encoded fashion on a substrate.
 43. The method of 41, wherein probes are immobilized on encoded microparticles which are in turn assembled in a random encoded array on a substrate.
 44. The method of 41, in which the calling step involves the use of a polymerase capable of extending or elongating probes whose interrogation site composition matches that of the designated polymorphic site in the target, and only those probes, by addition of one or more nucleoside triphosphates, one of which is labeled so as to generate an optically deectable signature
 45. The method of claim 41, wherein an optical signature is attached to all available immobilized probes in the first step by annealing to these primers a fluorescently labeled target to form a fluorescent hybridization complex, and wherein the second step involves the use of a polymerase capable of extending or elongating probes displaying a terminal match, and only those probes, by addition of one or more nucleotide triphosphates to form an extended hybridization complex, and wherein extension products are identified by the stability of optical signatures under an increase in temperature to a value selected to exceed the melting temperature of any hybridization complex but not to exceed the melting temperature of any extended hybridization complex.
 46. The method of claim 45, wherein probes are immobilized on encoded microparticles and the change in optical signature is detected, and particle identity determined, by flow cytometry.
 47. The method of claim 45, wherein probes are immobilized on encoded microparticles which are arranged in random encoded arrays, said arrays optionally arranged in a spatially encoded manner, and the change in optical signature is detected, and particle identity is determined, by direct imaging.
 48. A method of sequence-specific amplification of assay signals produced in the analysis of a nucleic acid sequence of interest in a biological sample, the method permitting real-time monitoring of amplified signal, and comprising the following steps: (a) providing a temperature-controlled sample containment device with associated temperature control apparatus permitting real-time recording of optical assay signal produced within said device; (b) providing within said sample containment device a set of distinguishable, immobilized oligonucleotide probes capable forming a hybridization complex with the sequence of interest; (c) permitting the sequence to anneal to the set of immobilized oligonucleotide probes to form a hybridization complex; (d) contacting said hybridization complex with a polymerase to allow elongation of extension of the matched probes contained within a hybridization complex; (e) providing means to convert elongation or extension of matching probes into an optical assay signal; (f) providing an optical recording/imaging device capable of recording optical assay signals from the set of immobilized probes in real time; (g) performing one or more “annealing-extending-detecting-denaturing” cycles, each cycle increasing the number of extended or elongated probes in arithmetic progression and involving the following steps: (i) set a first temperature favoring the formation of a hybridization complex; (ii) set a second temperature favorable to polymerase-catalyzed extension; (iii) convert extension into optical signal; (iv) record/image optical signals/signatures from all immobilized probes; and (v) set a third temperature so as to ensure denaturation of all hybridization complexes. 